NF-AT polypeptides and polynucleotides and screening methods for immunosuppressive agents

ABSTRACT

The invention provides novel polypeptides which are associated with the transcription complex NF-AT, polynucleotides encoding such polypeptides, antibodies which are reactive with such polypeptides, polynucleotide hybridization probes and PCR amplification probes for detecting polynucleotides which encode such polypeptides, transgenes which encode such polypeptides, homologous targeting constructs that encode such polypeptides and/or homologously integrate in or near endogenous genes encoding such polypeptides, nonhuman transgenic animals which comprise functionally disrupted endogenous genes that normally encode such polypeptides, and transgenic nonhuman animals which comprise transgenes encoding such polypeptides. The invention also provides methods for detecting T cells (including activated T cells) in a cellular sample, methods for treating hyperactive or hypoactive T cell conditions, methods for screening for immunomodulatory agents, methods for diagnostic staging of lymphocyte differentiation, methods for producing NF-AT proteins for use as research or diagnostic reagents, methods for producing antibodies reactive with the novel polypeptides, and methods for producing transgenic nonhuman animals. Also included are methods and agents for activation of NF-AT dependent transcription, including agents which interfere with the production, modification of nuclear or cytoplasmic subunits, or the nuclear import of the cytoplasmic subunits. In particular, screening tests for novel immunosuppressants are provided based upon the ability of NF-AT to activate transcription.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/232,346,entitled “NF-AT Polypeptides and Polynucleotides and Screening Methodsfor Immunosuppressive Agents”, filed on Jan. 15, 1999, now U.S. Pat. No.6,352,830, which is a continuation-in-part of application Ser. No.08/507,032, entitled “Screening Methods for Immunosuppressive Agents”,filed on 31 Jul. 1995, now U.S. Pat. No. 5,989,810, which is afile-wrapper-continuation of application Ser. No. 08/228,944, filed onApr. 18, 1994, now abandoned, which is a file-wrapper-continuation ofapplication Ser. No. 07/749,385, filed on Aug. 22, 1991; now abandoned,and a continuation-in-part of application Ser. No. 08/260,174, entitled“NF-AT Polypeptides and Polynucleotides”, filed Jun. 13, 1994, now U.S.Pat. No. 6,197,925, which is a continuation-in-part of application Ser.No. 08/124,981, entitled “NF-AT Polypeptides and Polynucleotides”, filedSep. 20, 1993 (U.S. Pat. No. 5,837,840). These applications are herebyincorporated by reference herein.

STATEMENT OF RIGHTS

This invention was made in the course of work supported by the U.S.Government. The U.S. Government has therefore certain rights in thisinvention.

FIELD OF THE INVENTION

The invention provides novel polypeptides which are associated with thetranscription complex NF-AT, polynucleotides encoding such polypeptides,antibodies which are reactive with such polypeptides, polynucleotidehybridization probes and PCR amplification probes for detectingpolynucleotides which encode such polypeptides, transgenes which encodesuch polypeptides, homologous targeting constructs that encode suchpolypeptides and/or homologously integrate in or near endogenous genesencoding such polypeptides, nonhuman transgenic animals which comprisefunctionally disrupted endogenous genes that normally encode suchpolypeptides, and transgenic nonhuman animals which comprise transgenesencoding such polypeptides. The invention also provides methods fordetecting T cells (including activated T cells) in a cellular sample,methods for treating hyperactive or hypoactive T cell conditions,methods for screening for immunomodulatory agents, methods fordiagnostic staging of lymphocyte differentiation, methods for producingNF-AT proteins for use as research or diagnostic reagents, methods forproducing antibodies reactive with the novel polypeptides, and methodsfor producing transgenic nonhuman animals.

BACKGROUND OF THE INVENTION

The immune response is coordinated by the actions of cytokines producedfrom activated T lymphocytes, such as lymphocytes contacted withantigens. Cytokines are responsible for the control of proliferation andcell fate decisions among precursors of B cells, granulocytes andmacrophages. T lymphocytes having a broad spectrum of antigen receptorsare produced in the thymus as a product of the processes of selectionand differentiation. When these T cells migrate to the peripherallymphoid organs and encounter antigen, they undergo activation, duringthe process of which they produce large numbers of cytokines that actupon other cells of the immune system to coordinate their behavior tobring about an effective immune response.

T lymphocyte activation involves the specific regulation of many genesfrom minutes after the antigen encounter until at least 10 days later. Tcells may also be activated by stimuli such as the combination of acalcium ionophore (e.g., ionomycin) and an activator of protein kinaseC, such as phorbol myristate acetate (PMA). Several lectins, includingphytohemagglutinin (PHA) may also be used to activate T cells (Nowell,P. C. (1990) Cancer Res. 20:462-466). The T cell activation genes areroughly grouped based on the time after stimulation at which each geneis regulated. Early genes trigger the regulation of subsequent genes inthe activation pathway.

Because of the critical role of the T lymphocyte, agents that interferewith the early activation genes, such as cyclosporin A and FK506, areeffective immunosuppressants. These early activation genes are regulatedby transcription factors, such as NF-AT, that in turn are regulatedthrough interactions with the antigen receptor. These transcriptionfactors act through enhancer and promoter elements on the earlyactivation genes to modulate their rate of transcription.

A typical early gene enhancer element is located in the first 325 basepairs upstream of the start site of the interleukin-2 gene. This regionhas been used extensively to dissect the requirements for T lymphocyteactivation. This region binds an array of transcription factorsincluding NF-AT, NFkB, Ap-1, Oct-1, and a newly identified protein thatassociates with Oct-1 called OAP-40. These different transcriptionfactors act together to integrate the complex requirements for Tlymphocyte activation.

The interleukin-2 gene is essential for both the proliferation andimmunologic activation of T cells. The signaling pathways which connectthe IL-2 gene and a representative and important early gene with theantigen receptor on the T cell surface and the signal transmissionpathways between them are illustrated in FIG. 1. The binding site forthe NF-AT protein appears to restrict expression of the interleukin-2gene and other early activation genes to the context of an activated Tlymphocyte. This information is based upon past work by Durand et al.,Mol. and Cell. Biol. (1988), Shaw et al., Science, 241: 202 (1988), andVerwiej et al., (1990) J. Biol. Chem, 265: 15788-15795 (1990).

NF-AT appears to be the most important element among the group mentionedabove in that it is able to direct transcription of any genes toactivated T cells in the context of an intact transgenic animal (Verweijet al. J. Biol. Chem. 265:15788-15795, 1990). NF-AT is also the onlyelement that requires physiologic activation through the antigenreceptor for the activation of transcription by NF-AT. For example, theelement is activated only after proper presentation of antigen ofexactly the correct sequence by MHC-matched antigen presenting cells.This effect can be mimicked by pharmacologic agents, including thecombination of ionomycin and PMA, which can also activate T cellsthrough critical early genes.

Other elements within the IL-2 enhancer, for example, the NF-kB site orthe AP-1 site, activate transcription in response to less specificstimuli, such as tumor necrosis factor alpha or simply PMA by itself.These compounds do not activate the IL-2 gene and other early activationgenes and do not lead to T cell activation. Such observations have ledto the conclusion that NF-AT restricts the expression of certain earlygenes, such as the interleukin-2 gene to their proper biologic context.Preliminary data have also indicated that a selective genetic deficiencyof NF-AT produces severe combined immunodeficiency (SCID) (Chatilla, T.et al. New Engl. J. Med. 320:696-702, 1989).

As noted above, cyclosporin A (CsA) and FK506 are capable of acting asimmunosuppressants. These agents inhibit T and B cell activation, mastcell degranulation and other processes essential to an effective immuneresponse (Borel et al. (1976) Agents Actions 6: 468; Sung et al. (1988)J. Exp. Med. 168: 1539; Gao et al. Nature 336: 176). In T lymphocytes,these drugs disrupt an unknown step in the transmission of signals fromthe T cell antigen receptor to cytokine genes that coordinate the immuneresponse (Crabtree et al. (1989) Science 243: 355; Schreiber et al.(1989) Science 251:283; Hohman & Hutlsch (1990) New Biol. 2: 663).

Putative intracellular receptors for FK506 and CsA have been describedand found to be cis-trans prolyl isomerases (Fischer & Bang (1985)Biochim. Biophys. Acta 828: 39; Fischer et al. Nature 337: 476;Handschumacher et al. (1984) Science 226: 544; Lang & Schmid (1988)Nature 331: 453; Standaert et al. (1990) Nature 346: 671). Binding ofthe drugs inhibits isomerase activity; however, studies with otherprolyl isomerase inhibitors (Bierer et al. (1990) Science 250: 556) andanalysis of cyclosporin-resistant mutants in yeast suggest that theprevention of T lymphocyte activation results from formation of aninhibitory complex involving the drug and the isomerase (Bierer et al.(1990) Proc. Natl. Acad. Sci. U.S.A. 87: 9231; Tropschug et al. (1989)Nature 342: 953), and not from inhibition of the isomerase activity perse.

The transcription factor NF-AT appears to be a specific target ofcyclosporin A and FK506, since transcription directed by this protein iscompletely blocked in T cells treated with these drugs, with little orno effect on other transcription factors, such as AP-1 and NF-_(K)B(Shaw et al.(1988) op.cit; Emmel et al. (1989) Science 246: 1617;Mattila et al. (1990) EMBO J. 9: 4425). However, the drugs' actualmechanism of action remains unclear. Unfortunately, while both arepotent immunosuppressive agents, neither cyclosporin nor FK506 are idealdrugs.

For example, cyclosporin adverse reactions include renal dysfunction,tremors, nausea and hypertension. Indeed, for many years researchershave attempted to develop superior replacements, with FK506 being themost recent candidate. Without understanding how cyclosporin (or FK506)functions at the intracellular level, developing improvedimmunosuppressants represents an extremely difficult research effortwith a very limited likelihood of success.

Thus, there exists a significant need to understand the functional basisof cyclosporin and FK506 effectiveness. With such knowledge, improvedassays for screening drug candidates would become feasible, which couldin turn dramatically enhance the search process. The present inventionfulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides novel methods and compositions useful,e.g., in screening for immunosuppressive agents. The invention is basedin part on the discovery of the overall mechanism by which NF-AT isformed intracellularly from nuclear and cytoplasmic subunits and on theisolation of nucleic acids encoding NF-AT proteins.

A basis of the present invention is the discovery that NF-AT (i.e., acomplex comprising NF-AT_(c) and NF-AT_(n)) is formed when a signal fromthe antigen receptor induces a preexisting cytoplasmic NF-AT submit(NF-AT_(c)) to translocate to the nucleus and combine with a nuclearNF-AT subunit (NF-AT_(n)). Cyclosporin A and FK506 block translocationof the cytoplasmic component without affecting the nuclear subunit. Aplausible synthesis of these studies and previous work posits that theprolyl isomerases, FK506-binding protein (FK-BP) and cyclophilin, alsofunction to import proteins to the nucleus.

The invention is also based on the purification of two related proteins,NF-ATc and NF-ATp, encoded by separate genes that represent thepreexisting or cytosolic components of NF-AT. The carboxy-terminal halfof NF-AT_(c) shows limited similarity to the DNA binding anddimerization regions of the Dorsal/Rel family of transcription factors(FIG. 15, for review, Nolan and Baltimore (1992) Current Biology, Ltd.2: 211-220) however, NF-AT_(c) appears to be the most distantly relatedmember of the group. Expression of a full length cDNA for one of theseproteins, NF-AT_(c), activates the IL-2 promoter in non-T lymphocytes,while a dominant negative of NF-AT_(c) specifically blocks activation ofthe IL-2 promoter in T lymphocytes, indicating that NF-AT_(c) isrequired for IL-2 gene expression and is responsible for the restrictedexpression of IL-2. NF-AT_(c) RNA expression is largely restricted tolymphoid tissues and is induced upon cell activation. The secondprotein, NF-AT_(p), is highly homologous to NF-AT_(c) over a limiteddomain, but exhibits wider tissue distribution and is highly expressedin tissues characterized by Ca++-dependent regulation. Together theseproteins are members of a new family of DNA binding proteins, which aredistantly related to the Dorsal/Rel family (Nolan and Baltimore (1992)Current Biology, Ltd. 2: 211-220). Agents that increase intracellularCa++ or that activate protein kinase C independently produce alterationsin the mobility of NF-AT_(c), indicating that distinct signalingpathways converge on NF-AT_(c) to regulate its function.

In accordance with one aspect of the invention, novel compositionsinclude NF-ATc polypeptides, nuclear components of NF-AT complexes, e.g,an NF-AT_(n) polypeptide, mixtures of the polypeptides, and cellularextracts containing the polypeptides. The NF-AT_(n) and NF-AT_(c)subunits are capable of forming a native NF-AT complex which binds in asequence-specific manner to a transcriptional regulatory DNA sequence ofan immune response gene. The NF-AT_(n) subunit is characterized by:

-   -   i. a molecular weight of about 45 kd;    -   ii. inducible expression in T cells (such as Jurkat cells);    -   iii. inducible expression in HeLa cells by exposing the cells to        an agent (such as PMA) capable of activating protein kinase C;    -   iv. a lack of effect by cyclosporin and FK506 on NF-AT_(n)        synthesis in T cells; and    -   v. specifically binding to an NF-AT_(c).

The NF-AT_(c) subunit is characterized by:

-   -   i. a molecular weight of about 90 kd;    -   ii. constitutively expressed in T cells;    -   iii. ability to be transported into a nucleus after a Ca++ flux        in the cell;    -   iv. nuclear transport inhibited by cyclosporin and FK506; and    -   v. specifically binding to an NF-AT_(n).

In another aspect of the present invention, isolated or purified nucleicacid sequences (or their complementary sequences) are provided which arecapable of binding to an NFAT complex, wherein the sequences aresubstantially homologous to an enhancer, such as IL-2 and IL-4enhancers, particularly the sequence AAGAGGAAAAA (SEQ ID NO: 53).

In another aspect, the invention embraces methods of screening for animmune regulating agent comprising combining the agent with a componentselected from the group consisting of an NF-AT_(n) polypeptide, anNF-AT_(c) polypeptide, and mixtures thereof, and determining whether theagent binds to the selected component.

In another embodiment, candidate immunomodulatory agents are identifiedby their ability to block the binding of a NF-AT_(c) polypeptide toother components of NF-AT (e.g., AP-1) and/or to block the binding ofNF-AT to DNA having an NF-AT recognition site. The DNA preferablyincludes one or more NF-AT binding sites at which a NF-AT proteincomplex specifically binds. One means for detecting binding of a NF-ATprotein comprising NF-AT_(c) to DNA is to immobilize the DNA, such as bycovalent or noncovalent chemical linkage to a solid support, and tocontact the immobilized DNA with a NF-AT protein complex comprising aNF-AT_(c) polypeptide that has been labeled with a detectable marker(e.g., by incorporation of radiolabeled amino acid). Such contacting istypically performed in aqueous conditions which permit binding of aNF-AT protein to a target DNA containing a NF-AT binding sequence.Binding of the labeled NF-AT to the immobilized DNA is measured bydetermining the extent to which the labeled NF-AT_(c) polypeptide isimmobilized as a result of a specific binding interaction. Such specificbinding may be reversible, or may be optionally irreversible if across-linking agent is added in appropriate experimental conditions.

In yet another embodiment, methods of screening for an immune regulatingagent will comprise the steps of:

-   -   i. preparing a collection of eukaroytic cells containing        NF-AT_(c) in the cytoplasm of the cell;    -   ii. treating the cells with an agent;    -   iii. assaying for nuclear translocation of the NF-AT_(c) wherein        blocking of nuclear transport correlates with the        immunosuppressive activity of the agent. The step of assaying        for nuclear translocation preferably comprises determining the        nuclear presence of the NF-AT_(c) which is labeled with a        detectable marker. Alternatively, the step of assaying for        nuclear translocation comprises determining nuclear association        between the NF-AT_(c) and an NF-AT_(n), preferably using nuclei        treated previously with the agent.

The assaying step can also comprise determining binding of NF-AT to aDNA sequence in the cell, such as by determining mRNA transcriptionlevels in the cell, wherein the mRNA encodes an immune response gene.

In a different embodiment, the method of screening for immune regulatingagents can comprise:

-   -   i. constructing a chimeric gene comprising an NF-AT regulated        enhancer region linked to a reporter gene (e.g., chloramphenicol        acetyltransterase (CAT) gene);    -   ii. inserting the chimeric gene into T cells;    -   iii. treating the T cells with T cell activating compounds in        the presence or absence of the agent; and    -   iv. determining the effect of the agent on expression of the        reporter gene.        Inhibition of expression of the reporter gene indicates that the        agent is a candidate immunosuppressant agent.

In one aspect, candidate immunomodulatory agents are identified as beingagents capable of inhibiting (or enhancing) intermolecular bindingbetween NF-AT_(c) and other polypeptides which comprises a NF-AT complex(e.g., AP-1, JunB, etc.). The invention provides methods andcompositions for screening libraires of agents for the capacity tointerfere with binding of NF-AT_(c) to other NF-AT polypeptide speciesunder aqueous binding conditions. Typically, at least either NF-AT_(c)and/or another NF-AT polypeptide species is labeled with a detectablelabel and intermolecular binding between NF-AT_(c) and other NF-ATpolypeptide species is detected by the amount of labeled speciescaptured in NF-AT complexes and the like.

For example, methods of assaying for a candidate-immunosuppressant agentcomprise mixing the agent with NF-AT_(n) and NF-AT_(C) under conditionswhich permit specific multimerization to form NF-AT, comprisingdimerization of NF-AT_(n) and NF-AT_(c), and determining whether saiddimerization (and/or multimerization with other subunits) occurs.Typically, NF-AT_(n) or NF-AT_(c), is immobilized and at least onesubunit is labeled with a detectable marker, most usually thenon-immobilized NF-AT subunit is labeled.

The present invention further provides several novel methods andcompositions for modulating the immune response and for screening formodulators of the immune response which utilize polynucleotide sequencesencoding NF-AT_(c) recombinant proteins and complementarypolynucleotides which are substantially identical to NF-AT_(c)polynucleotide sequences.

Thus, in another aspect of the invention, NF-AT_(c) polypeptidescomprising polypeptide sequences which are substantially identical to asequence shown in FIG. 12 or a cognate NF-AT_(c) amino acid sequence areprovided.

The invention also provides for nucleic acid sequences encodingNF-AT_(c). The characteristics of the cloned sequences are given,including the nucleotide and predicted amino acid sequence in FIG. 12.Polynucleotides comprising these sequences can serve as templates forthe recombinant expression of quantities of NF-AT_(c) polypeptides, suchas human NF-AT_(c) and murine NF-AT_(c). Polynucleotides comprisingthese sequences can also serve as probes for nucleic acid hybridizationto detect the transcription and mRNA abundance of NF-AT_(c) mRNA inindividual lymphocytes (or other cell types) by in situ hybridization,and in specific lymphocyte populations by Northern blot analysis and/orby in situ hybridization (Alwine et al. (1977) Proc. Natl. Acad. Sci.U.S.A. 74: 5350) and/or PCR amplification and/or LCR detection. Suchrecombinant polypeptides and nucleic acid hybridization probes haveutility for in vitro screening methods for immunomodulatory agents andfor diagnosis and treatment of pathological conditions and geneticdiseases, such as transplant rejection reactions, T cell-mediated immuneresponses, lymphocytic leukemias (e.g., T cell leukemia or lymphoma)wherein NF-AT activity contributes to disease processes, autoimmunedisease, arthritis, and the like.

The invention also provides antisense polynucleotides complementary toNF-AT_(c) sequences which are employed to inhibit transcription and/ortranslation of the cognate mRNA species and thereby effect a reductionin the amount of the respective NF-AT_(c) protein in a cell (e.g., a Tlymphocyte of a patient). Such antisense polynucleotides can function asimmunomodulatory drugs by inhibiting the formation of NF-AT proteinrequired for T cell activation.

In a variation of the invention, polynucleotides of the invention areemployed for diagnosis of pathological conditions or genetic diseasethat involve T cell neoplasms or T cell hyperfunction of hypofunction,and more specifically conditions and diseases that involve alterationsin the structure or abundance of NF-AT_(c) polypeptide, NF-AT_(c)polynucleotide sequence, or structure of the NF-AT_(c) gene or flankingregion(s).

The invention also provides antibodies which bind to NF-AT_(c) with anaffinity of about at least 1×10⁷ M⁻¹ and which lack specific highaffinity binding for other proteins present in activated T cells. Suchantibodies can be used as diagnostic reagents to identify T cells (e.g.,activatable T cells) in a cellular sample from a patient (e.g., alymphocyte sample, a solid tissue biopsy) as being cells which containan increased amount of NF-AT_(c) protein determined by standardizationof the assay to be diagnostic for activated T cells. Frequently,anti-NF-AT_(c) antibodies are included as diagnostic reagents forimmunohistopathology staining of cellular samples in situ. Additionally,anti-NF-AT_(c) antibodies may be used therapeutically by targeteddelivery to T cells (e.g., by cationization or byliposome/immunoliposome delivery).

The invention also provides NF-AT_(c) polynucleotide probes fordiagnosis of neoplasia or immune status by detection of NF-AT_(c) mRNAin cells explanted from a patient, or detection of a pathognomonicNF-AT_(c) allele (e.g., by RFLP or allele-specific PCR analysis). Apathognomonic NF-AT_(c) allele is an allele which is statisticallycorrelated with the presence of a predetermined disease or propensity todevelop a disease. Typically, the detection will be by in situhybridization using a labeled (e.g., ³²P, ³⁵S, ¹⁴C, ³H, fluorescent,biotinylated, digoxigeninylated) NF-AT_(c) polynucleotide, althoughNorthern blotting, dot blotting, or solution hybridization on bulk RNAor poly A⁺ RNA isolated from a cell sample may be used, as may PCRamplification using NF-AT_(c)-specific primers. Cells which contain anincreased amount of NF-AT_(c) mRNA as compared to standard controlvalues for cells or cell types other than activated T cells oractivatable T cells will be thereby identified as activated T cells oractivatable T cells. Similarly, the detection of pathognomonicrearrangements or amplification of the NF-AT_(c).locus or closely linkedloci in a cell sample will identify the presence of a pathologicalcondition or a predisposition to developing a pathological condition(e.g., cancer, genetic disease).

The present invention also provides a method for diagnosing T cellhypofunction of hyperfunction in a human patient, wherein a diagnosticassay (e.g., immunohistochemical staining of fixed lymphocytic cells byan antibody that specifically binds human NF-AT_(c)) is used todetermine if a predetermined pathognomonic concentration of NF-AT_(c)protein or NF-AT_(c) mRNA is present in a biological sample from a humanpatient; if the assay indicates the presence of NF-AT_(c) protein orNF-AT_(c) mRNA at or above such predetermined pathognomonicconcentration, the patient is diagnosed as having T cell hyperfunctionor hypofunction condition, or transplant rejection and the like.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Representation of the signal transmission pathways carryinginformation from the T lymphocyte antigen receptor to the earlyactivation genes that lead to proliferation, clonal expansion and immunefunction or to cell death (apoptosis), clonal deletion and tolerance.Primary signals emanate from the interaction of the T cell antigenreceptor (includes the TCR and CD3 complex) with antigen bound by themajor histocompatibility complex (MHC). Accessory signaling moleculessuch as CD2, CD4, and LFA-1 augment the primary signal. A secondarysignal that is required to completely activate T lymphocytes is providedby interleukins 1 and 6. These initial signals are transmitted to thenucleus by second messengers such as tyrosine phosphatases (CD-45),tyrosine kinases (lck and fyn), as well as by protein kinase C (PKC) andintracellular calcium. As depicted in the schematic, immunosuppressivedrugs such as FK506 and cyclosporin (CsA) as well as immune deficiencydiseases (SCID) interfere with the proper transmission of signals fromthe TCR to the nucleus.

FIG. 2. Diagram of the human IL-2 enhancer from −326 to +47 base pairs.DNase I protected regions are noted by boxes along with theidentification of the sites (A-E) and the name(s) of the proteins whichcomplex with these sites. Mutations introduced in the boxed regionsdrastically reduce IL-2 transcription following T lymphocyte activationand are indicated as percent wild type (full) expression remaining. Thearrow identifies the transcriptional start site.

FIG. 3. The diagram shows the NF-AT binding site from the distal elementin the interleukin-2 gene. Contact guanine residues are indicated bylower case letters and the construction of binding sites used to measureNF-AT dependent transcriptional activity are shown as an array of threeNF-AT binding sites.

FIG. 4. NF-AT is T cell enriched and is formed following activation of Tlymphocytes. Representation of NF-AT in different cell lines. Nuclearextracts from: J, Jurkat cells; K, KB cells (a derivative of HeLacells); F, Faza cells (a rat liver cell line); H, Hep G2 cells (a humanhepatocyte line); T, TEPC murine B-cell line; E, EL-4 murine T cellline; C, C2Cl2 murine myoblasts. Lanes labelled “+” are the complexesformed with nuclear extracts from cells treated with PHA (2 μg/ml) andPMA (50 ng/ml) for two hours.

FIG. 5. The nuclear component of the NF-AT complex requires proteinsynthesis while the cytoplasmic component of NF-AT is pre-existing in Tcells. Lanes 1 and 2, gel mobility shift assay using nuclear extracts(10 μg) from stimulated/FK5060-treated (s+F) cells in the presence (+)and absence (−) of 100 μM anisomycin. Lanes 3-6, complementation ofnuclear extracts from stimulated/FK506-treated cells (with or withoutanisomycin pretreatment) with cytoplasmic extracts prepared fromnonstimulated cells treated with or without anisomycin. Arrows indicatethe mobility of the reconstituted NF-AT DNA binding complex.

FIG. 6. NF-AT binding activity can be quantitatively reconstituted fromnuclear and cytoplasmic fractions of stimulated/FK506- andstimulated/CsA-treated Jurkat cells. (a) In lanes 1-6, nuclear extracts(10 μg) and cytoplasmic extracts (10 μg) from nonstimulated (ns),stimulated (s) with PMA/ionomycin, and stimulated/FK506-treated (s+F)cells were tested for NF-AT binding activity using electrophoretic gelmobility shift assays. In lanes 7-10, NF-AT was reconstituted by mixingnuclear and cytoplasmic extracts. Stimulated/FK506-treated (s+F) nuclearextracts (5 μg) were complemented with cytoplasmic extracts (5 μg) from:lane 7, nonstimulated (ns); lane 8, stimulated (s); lane 9,stimulated/FK506-treated (s+F); and lane 10,stimulated/rapamycin-treated (rap) cells. In all cases arrows indicatethe NF-AT protein DNA complex. (b) In lanes 1-3, mixing of nuclearextracts from nonstimulated cells (5 μg) with any cytoplasmic extracts(5 μg) fails to reconstitute NF-AT binding. In lanes, 4-9, reconstitutedNF-AT binding activity demonstrates DNA binding specificity: nuclearextracts (5 μg) from stimulated/FK506-treated cells (s+F) were mixedwith cytoplasmic extracts (5 μg) and competition was carried out with 10ng of unlabeled NF-AT or mutant NF-AT oligonucleotide. (c) The effect ofFK506 was tested on a murine T cell hybridoma, JK12/90.1 (Karttunen etal. (1991) PNAS 88:3972). In lanes 1-3, nuclear extracts (10 μg) fromnonstimulated/FK506-treated (s+F) Jurkat cells were tested for NF-ATbinding activity. Lane 6 shows NF-AT binding in nuclear extracts fromstimulated/FK506treated (s+F) JK12/90.1 cells. In lanes 4-5 and 7-8,NF-AT is reconstituted by mixing nuclear extracts (5 μg) fromstimulated/FK506-treated Jurkat or JK12/90.1 cells with cytoplasmicextracts (5 μg) from nonstimulated Jurkat or JK12/90.1 cells. (d) Inlanes 1-3, nuclear extracts (5 μg) from nonstimulated (ns), stimulated(s), and stimulated/cyclosporin A-treated (s+C) cells.Stimulated/cyclosporin A-treated (s+C) nuclear extracts (5 μg) werecomplemented with cytoplasmic fractions (5 μg) from: lane 4,nonstimulated (ns); lane 5, stimulated (s); and lane 6,stimulated/cyclosporin A-treated (s+C) cells.

FIG. 7. The nuclear component of NF-AT is present in HeLa cells and canbe complemented by Jurkat cytoplasm, but not by HeLa cell cytoplasm, toreconstitute NF-AT binding activity. (a) Lanes 1-6, gel mobility shiftassay using HeLa nuclear (10 μg) and cytoplasmic (10 μg) extracts fromnonstimulated (ns), stimulated (s), and stimulated/FK506-treated (s+F)cells do not form a NF-AT protein-DNA complex. In lanes 7-9, homologousmixing of nuclear extracts (5 μg) from stimulated/FK506-treated (s+F)HeLa cells with HeLa cytoplasmic extracts (5 μg) does not reconstituteNF-AT. (b) NF-AT binding activity is reconstituted with HeLa nuclear andJurkat cytoplasmic extracts. Nuclear extracts (5 μg) fromstimulated/FK506 treated HeLa cells complemented by cytoplasmic extracts(5 μ) from: lane 1, nonstimulated (ns); and lane 3,stimulated/FK506-treated (s+F) Jurkat cells. In lanes 4-9, reconstitutedNF-AT binding complex demonstrates DNA binding specificity when competedby 10 μg of unlabeled NF-AT or mutant NF-AT oligonucleotides. (c) Lanes1-3, heterologous mixing of Jurkat nuclear extracts (5 μg) fromstimulated/FK506 treated (s+F) with HeLa-cytoplasmic extracts (5 μg)does not reconstitute NF-AT binding activity.

FIG. 8. The nuclear component of NF-AT is induced by PMA while calciummediated signals allow translocation of the preexisting cytoplasmicsubunit of NF-AT. (a) Lanes 1 and 2, gel mobility shift assay usingnuclear extracts (10 μg) from PMAstimulated (p) and ionomycin-stimulated(i) cells. In lanes 3-8, complementation of nuclear extracts (5 μg) fromPMAstimulated and ionomycin-stimulated cells with cytoplasmic extracts(5 μg) from nonstimulated (ns), stimulated (s), andstimulated/FK506-treated (s+F) cells. Stimulated/FK506-treated (s+F)nuclear extracts (5 μg) were complemented with cytoplasmic extracts (5μg) from: lane 9, nonstimulated (ns); lane 10, PMA-stimulated (p); lane11, ionomycin-stimulated (i); and lane 12, stimulated (s) cells. Arrowsindicate the NF-AT protein DNA binding complex.

FIG. 9. In vitro transcription directed by the IL-2 enhancer or threetandemly linked NF-AT binding sites in nuclear extracts stimulated underdifferent conditions. (a) IL-2 directed transcription: lane 1,nonstimulated; lane 2, PMA/ionomycin/FK506-treated cells; andPMA/ionomycin stimulated cells, lane 3. NF-AT directed transcription:lane 4,cdescriptionde nonstimulated; lane 5, PMA/ionomycin/FK506 treatedcells; lane 6, PMA/ionomycin-stimulated cells. Expression from the IL-2enhancer and NF-AT G-less template generates a 401 and 383 nucleotide(nt) transcript, respectively. The adenovirus major late promoter(AdMLP) internal control generates a 28Q nt transcript. Fold inductionis calculated following normalization to AdMLP transcription. (b)Ribonuclease protection assay of NF-AT driven lac-Z mRNA. Lane 1,nonstimulated; lane 2, PMA/ionomycin-stimulated; lane 3,PMA/ionomycin/FK506-treated cells.

FIG. 10. NF-AT dependent T-antigen transcription levels in tissues oftransgenic mice. Total RNA was prepared from tissues of a 6-week-oldmouse of line Tag8 (Verweij et al. JBC 265:15788-15795 (1990)). Spleen,thymus and bone marrow cells were cultured for 24 hours in the presenceof ionomycin (0.6 μM1 and PMA (10 μg/ml). Ten micrograms of each RNAsample was used in an RNase protection assay. As a probe we used the 176nucleotide P-32 labeled antisense NF-AT-Tag RNA probe. Correctlyinitiated mRNA would yield a 47-nucleotide protected fragment. Theposition of the fragment (TI) is indicated by an arrow.

FIG. 11. Dephosphorylation of NF-AT inhibits its DNA binding. Lanes 1-5,gel mobility shift assay. Nuclear extracts (10 μg) from PMA/ionomycinstimulated Jurkat cells were incubated with several protein phosphataseinhibitors in the presence or absence of calf intestinal phosphatase.Characteristic NF-AT mobility shift in the presence of: lane 1, 200 μmsodium vanadate (Na₂VO₄); lane 2, 200 mM sodium molybdate (Na₂MO₄); lane3, 10 mM sodium fluoride (NaF); lane 4, one unit of calf intestinalphosphatase (CIP); lane 5, one unit of calf intestinal phosphatase plus200 μM of sodium vanadate, 200 μM sodium molybdate and 10 mm sodiumfluoride. For methods see FIG. 6. The arrow indicates the NF-AT proteinDNA complex.

FIG. 12 Panels A-G (SEQ ID NOs: 45-46) shows the nucleotide sequence ofthe human NF-AT_(c) cDNA and the deduced amino acid sequence. Nindicates that a sequence ambiguity is present.

FIG. 13 shows the expression of NF-AT_(c) protein in T cells (Jurkat)and non-T cells (Cos).

FIG. 14 Panels A and B show that the NF-AT_(c) cDNA clone encodes aprotein that activates transcription from an NF-AT site and is capableof activating the IL-2 promoter in non-T cells.

FIG. 15 (SEQ ID Nos: 47-52) shows the homology between NF-AT_(c),NF-AT_(p), and Rel family members. The protein sequences of murineNF-AT_(p) and the Rel proteins Dorsal (the Drosophila axis-determiningprotein), human c-Rel, NF-KB p50, and NF-KB p65 are aligned to thesequence of NF-AT_(c). Numbering is with respect to NF-AT_(c). Identityto NF-AT_(c), open boxes; similarity in known residue function orstructure, shaded areas. Stars indicate regions in which NF-AT_(c)has: 1) a charge reversal relative to the majority of other Relproteins, or has 2) replaced a potential salt bridge residue with ahistidine or other chelating residue. Lower portion shows a schematic ofNF-AT_(c) and NF-AT_(p).

FIG. 16 (panels A-C). Panel A: Ribonuclease protection for humanNF-AT_(c) with RNA from Jurkat cells (lanes 1-6) or Hela cells (lane 7).The expected specific ribonuclease-resistant fragment is 304 nucleotides(arrow). Hela cells were non-stimulated. Jurkat cells were eithernon-stimulated or stimulated with 20 ng/ml PMA and 2 uM ionomycin for 3hours, plus or minus 100 ng/ml CsA added at the indicated times afterstimulation. Panel B: RNA from the following human cells: KJ (preB cellALL), JD-1 (B cell lineage ALL), K562 (erythroleukemia cell line), CML(bone marrow cells from a patient with a myeloid leukemia), human muscletissue, Hep G2 (liver cell line), HPB ALL (T cell line, nonstimulated orstimulated with 2 ug/ml PHA and 50 ng/ml PMA for 30 minutes), and Helacells analyzed by ribonuclease protection. A longer exposure of this gelindicates that the K562 cell line contains a small amount of NF-AT_(c)transcript. Panel C: NF-AT_(c) (upper panel) and NF-AT_(p) (lower panel)mRNA expression in mouse tissues and a skin tumor derived from NF-AT-Tagtransgenic mice (Verweij et al. (1990) J. Biol. Chem 265: 15788-15795).Cells were either non-stimulated or stimulated with 20 ng/ml PMA and 2uM ionomycin for 3 hours. RNA was measured by quantiative ribonucleaseprotection using murine cDNA probes. The predicted size of the fragmenthomologous to the probe is indicated by the arrows.

FIG. 17 (panels A-D). Panel A: Cos cells and Jurkat cells weretransfected with reporter constructs for NF-AT or HNF-1 (β28).Co-transfected expression vectors for NF-AT_(c) (+NF-AT) orHNF-1a(+HNF-1) were included where indicated, otherwise empty pBJ5vector was included. Cells were stimulated as indicated: PMA, P+I (PMAplus ionomycin). Panel B: Cos cells were transfected with IL-2luciferase and with expression vectors as in A. Stimulations were as inA. Data in A and B are expressed as fold induction of luciferaseactivity over nonstimulated value with empty pBJ5 vector. Bars representmean and range of 2-3 independent transfections. Panel C: Expression ofNF-AT_(c) in Cos cells gives rise to specific DNA binding activity. Gelmobility shifts using nuclear extracts from Cos cells transfected withpBJ5 (lanes 1 and 3), with NF-AT_(c) (lanes 2 and 4-7), fromnon-transfected Jurkat cells (lanes 8-11) or using cytosols from pBJ5-or NF-AT_(c)-transfected Cos cells (lanes 12-13, 15-16) combined withHela nuclear extract (lanes 15-16). Lane 14, Hela nuclear extract alone.Labeled AP-1 (lanes 1-2) or NF-AT (lanes 3-16) probes and coldcompetitor oligonucleotides are indicated. Arrows indicate specific AP-1and NF-AT complexes. Panel D: Antisera induced supershift of NF-AT.NF-AT and AP-1 gel mobility shifts using nuclear extracts fromstimulated Jurkat cells or murine thymocytes. Either no antisera,preimmune, or one of two different immune antisera was included asindicated. Arrows indicate specific NF-AT or AP1 complexes orsupershifted NF-AT complexes (*).

FIG. 18 shows dominant-negative NF-AT_(c). Jurkat Tag cells weretransfected with vector plasmid (control) or with the dominant negativeNF-AT_(c) plasmid, plus the indicated secreted alkaline phosphatasereporter plasmid. Transfected cells were transferred to fresh culturemedium 24 hours after transfection and secreted alkaline phosphataseactivity was measured (Clipstone and Crabtree (1992) Nature 357:695-698) 16 to 24 hours later, after stimulation with 1 uM ionomycinplus 20 ng/ml PMA (NF-AT and IL-2 reporters), 20 ng/ml PMA alone (AP-1reporter) or no stimulation (RSV reporter). Bars indicate, secretedalkaline phosphatase activity from cells transfected with the dominantnegative NF-AT_(c) as a percentage of the activity from cellstransfected in parallel with control plasmid, and represent dataobtained from (n) independent transfections. The dominant negativeNF-AT_(c) consists of a carboxy terminal truncation of the epitopetagged NF-AT_(c) expression plasmid extending to the PvuII site at aminoacid 463.

FIG. 19 shows changes in mobility of epitope tagged NF-AT_(c) expressedin Jurkat cells. Cells were transfected with NF-AT_(c) as in FIG. 13 andstimulated as shown for 2 hrs plus or minus 100 ng/ml CsA. Whole celllysates were analyzed by western blotting as in FIG. 13.

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to means of modulating transcription thatis dependent upon the presence of a linked cis-acting NF-AT site as wellas methods of causing and preventing formation of transcriptionallyactive NF-AT complexes, controlling expression of the early T lymphocyteactivation genes, and controlling transcription of the humanimmunodeficiency virus. The invention also relates to the formation ofactive NF-AT from nuclear and cytoplasmic subunits by a novel mechanism;control of induction of the nuclear precursor of NF-AT, as well ascontrol of the nuclear import of the cytoplasmic precursor of NF-AT,methods by which the nuclear import of NF-AT can be modulated andmethods by which the induction of the nuclear subunit of NF-AT can beprevented or enhanced. The methods of this invention are useful indetermining or controlling the expression of early T lymphocyteactivation genes as well as determining or controlling the expression ofselected constitutive genes that can be advantageously expressed in Tlymphocytes. In addition, the invention also pertains to the developmentof screening assays for agents that modulate the nuclear import of thecytoplasmic subunit of NF-AT or the induction of the nuclear subunit ofNF-AT, such agents are thereby identified as candidate immunosuppressantagents.

A basis of the present invention is the experimental finding thatmultimeric-complexes are formed when a signal from the antigen receptorinduces a pre-existing cytoplasmic subunit to translocate to the nucleusand combine with a newly synthesized nuclear subunit of NF-AT. Formationof a functional multimeric complex, which includes [NF-AT_(c):NF-AT_(n)]heterodimer, then facilitates transcriptional enhancement by interactingwith specific NF-AT recognition sequences near particular structuralgenes, such as the IL-2 gene. Since transcriptional enhancement of earlygenes, such as the IL-2 gene, is a critical step in the process of Tlymphocyte activation, candidate immunosuppressants can be identified byscreening for agents which interfere with the formation of functionalNF-AT heterodimer and/or inhibit transcriptional enhancement thatentails NF-AT interacting with specific NF-AT recognition sequences.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Generally, the nomenclatureused hereafter and the laboratory procedures in cell culture, moleculargenetics, and nucleic acid chemistry and hybridization described beloware those well known and commonly employed in the art. Standardtechniques are used for recombinant nucleic acid methods, polynucleotidesynthesis, and microbial culture and transformation (e.g.,electroporation, lipofection). Generally enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see, generally, Sambrook et al. Molecular Cloning. ALaboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference)which are provided throughout this document. The procedures therein arebelieved to be well known in the art and are provided for theconvenience of the reader. All the information contained therein isincorporated herein by reference.

Oligonucleotides can be synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Methods for PCR amplification are described in the art (PCR Technology:Principles and Applications for DNA Amplification ed. H A Erlich,Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guide to Methodsand Applications, eds. Innis, Gelfland, Snisky, and White, AcademicPress, San Diego, Calif. (1990); Mattila et al. (1991) Nucleic AcidsRes. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; and U.S. Pat. No. 4,683,202, which are incorporated herein byreference).

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described. Forpurposes of the present invention, the following terms are definedbelow.

1. Definitions

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage (Immunology—A Synthesis, 2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991), which is incorporated herein by reference).Stereoisomers (e.g., D-amino acids) of the twenty conventional aminoacids, unnatural amino acids such as α,α-disubstituted amino acids,N-alkyl amino acids, lactic acid, and other unconventional amino acidsmay also be suitable components for polypeptides of the presentinvention. Examples of unconventional amino acids include:4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine,ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine,3-methylhistidine, 5-hydroxylysine, ω-N-methylarginine, and othersimilar amino acids and imino acids (e.g., 4-hydroxyproline). In thepolypeptide notation used herein, the lefthand direction is the aminoterminal direction and the righthand direction is the carboxy-terminaldirection, in accordance with standard usage and convention. Similarly,unless specified otherwise, the lefthand end of single-strandedpolynucleotide sequences is the 5′ end; the lefthand direction ofdouble-stranded polynucleotide sequences is referred to as the 5′direction. The direction of 5′ to 3′ addition of nascent RNA transcriptsis referred to as the transcription direction; sequence regions on theDNA strand having the same sequence as the RNA and which are 5′ to the5′ end of the RNA transcript are referred to as “upstream sequences”;sequence regions on the DNA strand having the same sequence as the RNAand which are 3′ to the 3′ end of the RNA transcript are referred to as“downstream sequences”.

The term “transcriptional enhancement” is used herein to refer tofunctional property of producing an increase in the rate oftranscription of linked sequences that contain a functional promoter.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials such as bacteria, plants, fungi, or animal(particularly mammalian) cells or tissues. Agents are evaluated forpotential activity as immunosupressants by inclusion in screening assaysdescribed hereinbelow.

The terms “immunosuppressant” and “immunosuppressant agent” are usedherein interchangeably to refer to agents that have the functionalproperty of inhibiting an immune response in human, particularly animmune response that is mediated by activated T cells.

The terms “candidate immunosuppressant” and “candidate immunosuppressantagent” are used herein interchangeably to refer to an agent which isidentified by one or more screening method(s) of the invention as aputative inhibitor of T cell activation. Some candidateimmunosuppressants may have therapeutic potential.

The term “altered ability to modulate” is used herein to refer to thecapacity to either enhance transcription or inhibit transcription of agene; such enhancement or inhibition may be contingent on the occurrenceof a specific event, such as T cell stimulation. For example but not forlimitation, an agent that prevents expression of NF-AT_(C) protein willalter the ability of a T cell to modulate transcription of an IL-2 genein response to an antigen stimulus. This alteration will be manifest asan inhibition of the transcriptional enhancement of the IL-2 gene thatnormally ensues following T cell stimulation. The altered ability tomodulate transcriptional enhancement or inhibition may affect theinducible transcription of a gene, such as in the just-cited IL-2example, or may effect the basal level transcription of a gene, or both.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparision; a reference sequence may bea subset of a larger sequence, for example, as a segment of afull-length cDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of FIG. 1, or may comprise a complete cDNA orgene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2: 482, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.(U.S.A.) 85: 2444, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection, and the best alignment (i.e., resulting in thehighest percentage of homology over the comparison window) generated bythe various methods is selected. The term “sequence identity” means thattwo polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparision (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 85 percentsequence identity, preferably at least 90 to 95 percent sequenceidentity, more usually at least 99 percent sequence identity as comparedto a reference sequence over a comparison window of at least 20nucleotide positions, frequently over a window of at least 25-50nucleotides, wherein the percentage of sequence identity is calculatedby comparing the reference sequence to the polynucleotide sequence whichmay include deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. The reference sequencemay be a subset of a larger sequence, for example, as a segment of thefull-length human NF-AT_(c) polynucleotide sequence shown in FIG. 12 orthe full-length murine or bovine NF-AT_(c) cDNA sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term “NF-AT_(c) native protein” and “full-length NF-AT_(c) protein”as used herein refers to a a naturally-occurring NF-AT_(c) polypeptidecorresponding to the deduced amino acid sequence shown in FIG. 12 orcorresponding to the deduced amino acid sequence of a cognatefull-length cDNA. Also for example, a native NF-AT_(c) protein presentin naturally-occurring lymphocytes which express the NF-AT_(c) gene areconsidered full-length NF-AT_(c) proteins.

The term “NF-AT_(c) fragment” as used herein refers to a polypeptidethat has an amino-terminal and/or carboxy-terminal deletion, but wherethe remaining amino acid sequence is identical to the correspondingpositions in the NF-AT_(c) sequence deduced from a full-length cDNAsequence (e.g., the cDNA sequence shown in FIG. 1). NF-AT_(c) fragmentstypically are at least 14 amino acids long, preferably at least 20 aminoacids long, usually at least 50 amino acids long or longer.

The term “NF-AT_(c) analog” as used herein refers to polypeptides whichare comprised of a segment of at least 25 amino acids that hassubstantial identity to a portion of the deduced amino acid sequenceshown in FIG. 12, and which has at least one of the followingproperties: (1) binding to other NF-AT proteins (e.g., AP-1) undersuitable binding conditions, or (2) ability to localize to the nucleusupon T cell activation. Typically, NF-AT_(c) analog polypeptidescomprise a conservative amino acid substitution (or addition ordeletion) with respect to the naturally-occurring sequence. NF-AT_(c)analogs typically are at least 20 amino acids long, preferably at least50 amino acids long or longer, most usually being as long as full-lengthnaturally-occurring NF-AT_(c) (e.g., as shown in FIG. 12). SomeNF-AT_(c) analogs may lack biological activity but may still be employedfor various uses, such as for raising antibodies to NF-AT_(c) epitopes,as an immunological reagent to detect and/or purify α-NF-AT_(c)antibodies by affinity chromatography, or as a competitive ornoncompetitive agonist, antagonist, or partial agonist of nativeNF-AT_(c) protein function.

The term “NF-AT_(c) polypeptide” is used herein as a generic term torefer to native protein, fragments, or analogs of NF-AT_(c). Hence,native NF-AT_(c), fragments of NF-AT_(c), and analogs of NF-AT_(c) arespecies of the NF-AT_(c) polypeptide genus. Preferred NF-AT_(c)polypeptides include: the human full-length NF-AT_(c) protein comprisingthe polypeptide sequence shown in FIG. 12, or polypeptides consistingessentially of a sequence shown in Table II.

The term “cognate” as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example butnot limitation, in the human genome, the human CD4 gene is the cognategene to the mouse CD4 gene, since the sequences and structures of thesetwo genes indicate that they are highly homologous and both genes encodea protein which functions in signaling T cell activation through MHCclass II-restricted antigen recognition. Thus, the cognate murine geneto the human NF-AT_(c) gene is the murine gene which encodes anexpressed protein which has the greatest degree of sequence identity tothe human NF-AT_(c) protein and which exhibits an expression patternsimilar to that of the human NF-AT_(c) (e.g., expressed in T lineagecells). Preferred cognate NF-AT_(c) genes are: rat NF-AT_(c), rabbitNF-AT_(c), canine NF-AT_(c), nonhuman primate NF-AT_(c), porcineNF-AT_(c), bovine NF-AT_(c), and hamster NF-AT_(c).

The term “NF-AT_(c)-dependent gene” is used herein to refer to geneswhich: (1) have a NF-AT binding site (a site which can be specificallyfootprinted by NF-AT under suitable binding conditions) within about 10kilobases of the first coding sequence of said gene, and (2) manifest analtered rate of transcription, either increased or decreased, from amajor or minor transcriptional start site for said gene, wherein suchalteration in transcriptional rate correlates with the presence ofNF-AT_(c) polypeptide in NF-AT complexes, such as in an activated Tcell.

The term “candidate immunomodulatory agent” is used herein to refer toan agent which is identified by one or more screening method(s) of theinvention as a putative immunomodulatory agent. Some candidateimmunomodulatory agents may have therapeutic potential as drugs forhuman use.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), biotinyl groups, predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, metal binding domains, epitopetags). In some embodiments, labels are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual species in the composition), and preferably asubstantially purified fraction is a composition wherein the objectspecies comprises at least about 50 percent (on a molar basis) of allmacromolecular species present. Generally, a substantially purecomposition will comprise more than about 80 to 90 percent of allmacromolecular species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single macromolecularspecies.

As used herein the terms “pathognomonic concentration”, “pathognomonicamount”, and “pathognomonic staining pattern” refer to a concentration,amount, or localization pattern, respectively, of a NF-AT_(c) protein ormRNA in a sample, that indicates the presence of a hypofunctional orhyperfunctional T cell condition or a predisposition to developing adisease, such as graft rejection. A pathognomonic amount is an amount ofa NF-AT_(c) protein or NF-AT_(c) mRNA in a cell or cellular sample thatfalls outside the range of normal clinical values that is established byprospective and/or retrospective statistical clinical studies.Generally, an individual having a neoplastic disease (e.g., lymphocyticleukemia) or T cell-mediated immune response will exhibit an amount ofNF-AT_(c) protein or mRNA in a cell or tissue sample that is higher thanthe range of concentrations that characterize normal, undiseasedindividuals; typically the pathognomonic concentration is at least aboutone standard deviation above the mean normal value, more usually it isat least about two standard deviations or more above the mean normalvalue. However, essentially all clinical diagnostic tests produce somepercentage of false positives and false negatives. The sensitivity andselectivity of the diagnostic assay must be sufficient to satisfy thediagnostic objective and any relevant regulatory requirements. Ingeneral, the diagnostic methods of the invention are used to identifyindividuals as disease candidates, providing an additional parameter ina differential diagnosis of disease made by a competent healthprofessional.

2. NF-AT_(c) Polynucleotides

Genomic or cDNA clones encoding NF-AT_(c) may be isolated from clonelibraries (e.g., available from Clontech, Palo Alto, Calif.) usinghybridization probes designed on the basis of the nucleotide sequencesshown in FIG. 12 and using conventional hybridization screening methods(e.g., Benton W D and Davis R W (1977) Science 196: 180; Goodspeed etal. (1989) Gene 76: 1; Dunn et al. (1989) J. Biol. Chem. 264: 13057).Where a cDNA clone is desired, clone libraries containing cDNA derivedfrom T cell mRNA is preferred. Alternatively, synthetic polynucleotidesequences corresponding to all or part of the sequences shown in FIG. 12may be constructed by chemical synthesis of oligonucleotides.Additionally, polymerase chain reaction (PCR) using primers based on thesequence data disclosed in FIG. 12 may be used to amplify DNA fragmentsfrom genomic DNA, mRNA pools, or from cDNA clone libraries. U.S. Pat.Nos. 4,683,195 and 4,683,202 describe the PCR method. Additionally, PCRmethods employing one primer that is based on the sequence datadisclosed in FIG. 12 and a second primer that is not based on thatsequence data may be used. For example, a second primer that ishomologous to or complementary to a polyadenylation segment may be used.In an embodiment, a polynucleotide comprising the 2742 nucleotide-longsequence of FIG. 12 can be used. Alternative polynucleotides encodingthe 716 amino acid sequence of FIG. 12 can also be readily constructedby those of skill in the art by using the degeneracy of the geneticcode. Polynucleotides encoding amino acids 418 to 710 of the NF-AT_(c)sequence of FIG. 12 can also be constructed by those of skill in theart.

It is apparent to one of skill in the art that nucleotide substitutions,deletions, and additions may be incorporated into the polynucleotides ofthe invention. Nucleotide sequence variation may result from sequencepolymorphisms of various NF-AT_(c) alleles, minor sequencing errors, andthe like. However, such nucleotide substitutions, deletions, andadditions should not substantially disrupt the ability of thepolynucleotide to hybridize to one of the polynucleotide sequences shownin FIG. 12 under hybridization conditions that are sufficientlystringent to result in specific hybridization.

Specific hybridization is defined herein as the formation of hybridsbetween a probe polynucleotide (e.g., a polynucleotide of the inventionwhich may include substitutions, deletion, and/or additions) and aspecific target polynucleotide (e.g., a polynucleotide having thesequence in FIG. 12), wherein the probe preferentially hybridizes to thespecific target such that, for example, a single band corresponding toNF-AT_(c) mRNA (or bands corresponding to multiple alternative splicingproducts of the NF-AT_(c) gene) can be identified on a Northern blot ofRNA prepared from a suitable cell source (e.g., a T cell expressingNF-AT_(c)). Polynucleotides of the invention and recombinantly producedNF-AT_(c), and fragments or analogs thereof, may be prepared on thebasis of the sequence data provided in FIG. 12 according to methodsknown in the art and described in Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., (1989), Cold Spring Harbor, N.Y. and Bergerand Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.,which are incorporated herein by reference.

NF-AT_(c) polynucleotides may be short oligonucleotides (e.g., 25-100bases long), such as for use as hybridization probes and PCR (or LCR)primers. NF-AT_(c) polynucleotide sequences may also comprise part of alarger polynucleotide (e.g., a cloning vector comprising a NF-AT_(c)clone) and may be fused, by polynucleotide linkage, in frame withanother polynucleotide sequence encoding a different protein (e.g.,glutathione S-transferase or β-galactosidase) for encoding expression ofa fusion protein. Typically, NF-AT_(c) polynucleotides comprise at least25 consecutive nucleotides which are substantially identical to anaturally-occurring NF-AT_(c) sequence (e.g., FIG. 12), more usuallyNF-AT_(c) polynucleotides comprise at least 50 to 100 consecutivenucleotides which are substantially identical to a naturally-occurringNF-AT_(c) sequence. However, it will be recognized by those of skillthat the minimum length of a NF-AT_(c) polynucleotide required forspecific hybridization to a NF-AT_(c) target sequence will depend onseveral factors: G/C content, positioning of mismatched bases (if any),degree of uniqueness of the sequence as compared to the population oftarget polynucleotides, and chemical nature of the polynucleotide (e.g.,methylphosphonate backbone, phosphorothiolate, etc.), among others.

For example but not limitation, suitable hybridization probes fordetecting and/or quantifying the presence of NF-AT_(c) mRNA in a samplegenerally comprise at least one, preferably at least two, and morepreferably all of the following human NF-AT_(c) sequences shown in TableI, or their complements:

TABLE I Selected Human NF-AT_(c) Polynucleotide Sequences 5′-TTC CTC CGGGGC GCG CGG CGT GAG CCC GGG GCG AGG-3′; (SEQ ID NO: 1) 5′-CAG CGC GGGGCG GCC ACT TCT CCT GTG CCT CCG CCC GCT GCT-3′; (SEQ ID NO: 2) 5′-GCCGCG CGG ATG CCA AGC ACC AGC TTT CCA GTC CCT TCC AAG-3′; (SEQ ID NO: 3)5′-CCA ACG TCA GCC CCG CCC TGC CGC TCC CCA CGG CGC ACT CCA-3′; (SEQ IDNO: 4) 5′-TTC AGA CCT CCA CAC CGG GCA TCA TCC CGC CGG CGG-3′; (SEQ IDNO: 5) 5′-GCC ACA CCA GGC CTG ATG GGG CCC CTG CCC TGG AGA GTC CTC-3′;(SEQ ID NO: 6) 5′-AGT CTG CCC AGC CTG GAG GCC TAC AGA GAC CCC TCG TGCCTG-3′; (SEQ ID NO: 7) 5′-GTG TCT CCC AAG ACC ACG GAC CCC GAG GAG GGCTTT CCC-3′; (SEQ ID NO: 8) 5′-AGC TGG CTG GGT GCC CGC TCC TCC AGA CCCGCG TCC CCT TGC-3′; (SEQ ID NO: 9) 5′-TAC AGC CTC AAC GGC CGG CAG CCGCCC TAC TCA CCC CAC CAC-3′; (SEQ ID NO: 10) 5′-GAC CAC CGA CAG CAG CCTGGA CCT GGG AGA TGG CGT CCC TGT-3′; (SEQ ID NO: 11) 5′-CCT GGG CAG CCCCCC GCC CCC GGC CGA CTT CGC GCC CGA AGA-3′; (SEQ ID NO: 12) 5′-GCT CCCCTA CCA GTG GCG AAG CCC AAG CCC CTG TCC CCT ACG-3′; (SEQ ID NO: 13)5′-CTT CGG ATT GAG GTG CAG CCC AAG TCC CAC CAC CGA GCC CAC-3′; (SEQ IDNO: 14) 5′-CAT GGC TAC TTG GAG AAT GAG CCG CTG ATG CTG CAG CTT TTC-3′;(SEQ ID NO: 15) 5′-AAG ACC GTG TCC ACC ACC AGC CAC GAG GCT ATC CTC TCCAAC-3′; (SEQ ID NO: 16) 5′-TCA GCT CAG GAG CTG CCT CTG GTG GAG AAG CAGAGC ACG GAC-3′; (SEQ ID NO: 17) 5′-AAC GCC ATC TTT CTA ACC GTA AGC CGTGAA CAT GAG CGC G-3′; (SEQ ID NO: 18) 5′-AGA AAC GAC GTC GCC GTA AAG CAGCGT GGC GTG TGG CA-3′; and (SEQ ID NO: 19) 5′-GCA TAC TCA GAT AGT CACGGT TAT TTT GCT TCT TGC GAA TG-3′. (SEQ ID NO: 20)

Also for example but not limitation, the following pair of PCR primers(amplimers) may be used to amplify murine or human NF-AT_(c) sequences(e.g., by reverse transcriptase initiated PCR of RNA from NF-AT_(c)expressing cells):

(SEQ ID NO: 21) (forward) 5′-AGGGCGCGGGCACCGGGGCGCGGGCAGGGCTCGGAG-3′(SEQ ID NO: 22) (reverse) 5′-GCAAGAAGCAAAATAACCGTGACTATCTGAGTATGC-3′If desired, PCR amplimers for amplifying substantially full-length cDNAcopies may be selected at the discretion of the practioner. Similarly,amplimers to amplify single NF-AT_(c) exons or portions of the NF-AT_(c)gene (murine or human) may be selected.

Each of these sequences may be used as hybridization probes or PCRamplimers to detect the presence of NF-AT_(c) mRNA, for example todiagnose a disease characterized by the presence of an elevatedNF-AT_(c) mRNA level in lymphocytes, or to perform tissue typing (i.e.,identify tissues characterized by the expression of NF-AT_(c) mRNA), andthe like. The sequences may also be used for detecting genomic NF-AT_(c)gene sequences in a DNA sample, such as for forensic DNA analysis (e.g.,by RFLP analysis, PCR product length(s) distribution, etc.) or fordiagnosis of diseases characterized by amplification and/orrearrangements of the NF-AT_(c) gene.

Disclosure of the full coding sequence for human NF-AT_(c) shown in FIG.12 makes possible the construction of isolated polynucleotides that candirect the expression of NF-AT_(c), fragments thereof, or analogsthereof. Further, the sequences in FIG. 12 make possible theconstruction of nucleic acid hybridization probes and PCR primers thatcan be used to detect RNA and DNA sequences encoding NF-AT_(c).

Polynucleotides encoding full-length NF-AT_(c) or fragments or analogsthereof, may include sequences that facilitate transcription (expressionsequences) and translation of the coding sequences, such that theencoded polypeptide product is produced. Construction of suchpolynucleotides is well known in the art and is described further inManiatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. (1989),Cold Spring Harbor, N.Y. For example, but not for limitation, suchpolynucleotides can include a promoter, a transcription termination site(polyadenylation site in eukaryotic expression hosts), a ribosomebinding site, and, optionally, an enhancer for use in eukaryoticexpression hosts, and, optionally, sequences necessary for replicationof a vector. A typical eukaryotic expression cassette will include apolynucleotide sequence encoding a NF-AT_(c) polypeptide linkeddownstream (i.e., in translational reading frame orientation;polynucleotide linkage) of a promoter such as the HSV tk promoter or thepgk (phosphoglycerate kinase) promoter, optionally linked to an enhancerand a downstream polyadenylation site (e.g., an SV40 large T Ag poly Aaddition site).

A preferred NF-AT_(c) polynucleotide encodes a NF-AT_(c) polypeptidethat comprises at least one of the following amino acids sequences:

-NAIFLTVSREHERVGC-; (SEQ ID NO: 25) -LHGYLENEPLMLQLFIGT-; (SEQ ID NO:26) -PSTSPRASVTEESWLG-; (SEQ ID NO: 27) -GPAPRAGGTMKSAEEEHYG-; (SEQ IDNO: 28) -ASAGGHPIVQ-; (SEQ ID NO: 29) -NTRVRLVFRV-; (SEQ ID NO: 30)-AKTDRDLCKPNSLVVEIPPFRN-; (SEQ ID NO: 31) -EVQPKSHHRAHYETEGSR-; (SEQ IDNO: 32) -SPRVSVTDDSWLGNT-; (SEQ ID NO: 33) -SHHRAHYETEGSRGAV-; (SEQ IDNO: 34) -LRNSDIELRKGETDIGR-; and (SEQ ID NO: 35) -TLSLQVASNPIEC-. (SEQID NO: 36)The degeneracy of the genetic code gives a finite set of polynucleotidesequences encoding these amino acid sequences; this set of degeneratesequences may be readily generated by hand or by computer usingcommercially available software (Wisconsin Genetics Software PackageRelaes 7.0). Thus, isolated polynucleotides typically less thanapproximately 10,000 nucleotides in length and comprising sequencesencoding each of the following amino acid sequences:

-NAIFLTVSREHERVGC-; (SEQ ID NO: 25) -LHGYLENEPLMLQLFIGT-; (SEQ ID NO:26) -PSTSPRASVTEESWLG-; (SEQ ID NO: 27) -GPAPRAGGTMKSAEEEHYG-; (SEQ IDNO: 28) -ASAGGHPIVQ-; (SEQ ID NO: 29) -NTRVRLVFRV-; (SEQ ID NO: 30)-AKTDRDLCKPNSLVVEIPPFRN-; (SEQ ID NO: 31) -EVQPKSHHRAHYETEGSR-; (SEQ IDNO: 32) -SPRVSVTDDSWLGNT-; (SEQ ID NO: 33) -SHHRAHYETEGSRGAV-; (SEQ IDNO: 34) -LRNSDIELRKGETDIGR-; and (SEQ ID NO: 35) -TLSLQVASNPIEC-. (SEQID NO: 36)are provided and may be used for, among other uses, the expression of aNF-AT_(c) polypeptide which can be used as an immunogen, immunologicalreagent, and the like. Such polynucleotides typically comprise anoperably linked promoter for driving expression in a suitableprokaryotic or eukaryotic host cell. One exemplification of such apolynucleotide is the human NF-AT_(c) cDNA sequence of FIG. 12 cloned inoperable linkage to the mammalian expression vector pSRα, manyalternative embodiments will be apparent to those of skill in the art,including the use of alternative expression vectors (e.g., pBC12BI andp91023(B); Hanahan J (1983) J. Mol. Biol. 166: 577; Cullen et al. (1985)J. Virol. 53: 515; Lomedico P T (1982) Proc. Natl. Acad. Sci. (U.S.A.)79: 5798; Morinaga et al. (1984) Bio/Technology 2: 636).

Additionally, where expression of a polypeptide is not desired,polynucleotides of this invention need not encode a functional protein.Polynucleotides of this invention may serve as hybridization probesand/or PCR primers (amplimers) and/or LCR oligomers for detectingNF-AT_(c) RNA or DNA sequences.

Alternatively, polynucleotides of this invention may serve ashybridization probes or primers for detecting RNA or DNA sequences ofrelated genes, such genes may encode structurally or evolutionarilyrelated proteins. For such hybridization and PCR applications, thepolynucleotides of the invention need not encode a functionalpolypeptide. Thus, polynucleotides of the invention may containsubstantial deletions, additions, nucleotide substitutions and/ortranspositions, so long as specific hybridization or specificamplification to the NF-AT_(c) sequence is retained.

Specific hybridization is defined hereinbefore, and can be roughlysummarized as the formation of hybrids between a polynucleotide of theinvention (which may include substitutions, deletions, and/or additions)and a specific target polynucleotide such as human NF-AT_(c) mRNA sothat a single band is identified corresponding to each NF-AT_(c) isoformon a Northern blot of RNA prepared from T cells (i.e., hybridization andwashing conditions can be established that permit detection of discreteNF-AT_(c) mRNA band(s)). Thus, those of ordinary skill in the art canprepare polynucleotides of the invention, which may include substantialadditions, deletions, substitutions, or transpositions of nucleotidesequence as compared to sequences shown in FIG. 12 and determine whetherspecific hybridization is a property of the polynucleotide by performinga Northern blot using RNA prepared from a T lymphocyte cell line whichexpresses NF-AT_(c) mRNA and/or by hybridization to a NF-AT_(c) DNAclone (cDNA or genomic clone).

Specific amplification is defined as the ability of a set of PCRamplimers, when used together in a PCR reaction with a NF-AT_(c)polynucleotide, to produce substantially a single major amplificationproduct which corresponds to a NF-AT_(c) gene sequence or mRNA sequence.Generally, human genomic DNA or mRNA from NF-AT_(c) expressing humancells (e.g., Jurkat cell line) is used as the template DNA sample forthe PCR reaction. PCR amplimers that exhibit specific amplification aresuitable for quantitative determination of NF-AT_(c) mRNA byquantitative PCR amplification. NF-AT_(c) allele-specific amplificationproducts, although having sequence and/or length polymorphisms, areconsidered to constitute a single amplification product for purposes ofthis definition.

Generally, hybridization probes comprise approximately at least 25consecutive nucleotides of a sequence shown in FIG. 12 (for human andmurine NF-AT_(c) detection, respectively), preferably the hybridizationprobes contain at least 50 consecutive nucleotides of a sequence shownin FIG. 12, and more preferably comprise at least 100 consecutivenucleotides of a sequence shown in FIG. 12. PCR amplimers typicallycomprise approximately 25 to 50 consecutive nucleotides of a sequenceshown in FIG. 12, and usually consist essentially of approximately 25 to50 consecutive nucleotides of a sequence shown in FIG. 12 withadditional nucleotides, if present, generally being at the 5′ end so asnot to interfere with polymerase-mediated chain extension. PCR amplimerdesign and hybridization probe selection are well within the scope ofdiscretion of practioners of ordinary skill in the art.

In one preferred embodiment of the invention, hybridization probes thatspecifically identify the NF-AT_(c) gene may be used in methods fordiagnosing genetic disease. For example, but not for limitation, thegenetic disease thus diagnosed may involve a lesion in the relevantNF-AT_(c) structural or regulatory sequences, or may involve a lesion ina genetic locus closely linked to the NF-AT_(c) locus and which can beidentified by restriction fragment length polymorphism or DNA sequencepolymorphism at the linked NF-AT_(c) locus. In a further preferredembodiment, NF-AT_(c) gene probes are used to diagnose or identifygenetic disease involving predisposition to immunological disease,wherein the amount or functionality of endogenous NF-AT_(c) issufficient for the individual to exhibit an increased probability ofdeveloping an immune disease, particularly an immune deficiency,arthritis, or autoimmune disease.

3. Isolation of the Cognate Human NF-AT_(c) Gene

The human homolog of the NF-AT_(c) cDNA is identified and isolated byscreening a human genomic clone library, such as a human genomic libraryin yeast artificial chromosomes, cosmids, or bacteriophage λ (e.g., λCharon 35), with a polynucleotide probe comprising a sequence of aboutat least 24 contiguous nucleotides (or their complement) of the cDNAsequence shown in FIG. 12. Typically, hybridization and washingconditions are performed at high stringency according to conventionalhybridization procedures. Positive clones are isolated and sequenced.For illustration and not for limitation, a full-length polynucleotidecorresponding to the sequence of FIG. 12 may be labeled and used as ahybridization probe to isolate genomic clones from a human or murinegenomic clone libary in λEMBL4 or λGEM11 (Promega Corporation, Madison,Wis.); typical hybridization conditions for screening plaque lifts(Benton and Davis (1978) Science 196: 180) can be: 50% formamide, 5×SSCor SSPE, 1-5× Denhardt's solution, 0.1-1% SDS, 100-200 μg shearedheterologous DNA or tRNA, 0-10% dextran sulfate, 1×10⁵ to 1×10⁷ cpm/mlof denatured probe with a specific activity of about 1×10⁸ cpm/μg, andincubation at 42° C. for about 6-36 hours. Prehybridization conditionsare essentially identical except that probe is not included andincubation time is typically reduced. Washing conditions are typically1-3×SSC, 0.1-1% SDS, 50-70° C. with change of wash solution at about5-30 minutes.

Nonhuman NF-AT_(c) cDNAs and genomic clones (i.e., cognate nonhumanNF-AT_(c) genes) can be analogously isolated from various nonhuman cDNAand genomic clone libraries available in the art (e.g., Clontech, PaloAlto, Calif.) by using probes based on the sequences shown in FIG. 12,with hybridization and washing conditions typically being less stringentthan for isolation of human NF-AT_(c) clones.

Polynucleotides comprising sequences of approximately at least 30-50nucleotides, preferably at least 100 nucleotides, corresponding to orcomplementary to the nucleotide sequences shown in FIG. 12 can serve asPCR primers and/or hybridization probes for identifying and isolatinggermline genes corresponding to NF-AT_(c). These germline genes may behuman or may be from a related mammalian species, preferably rodents orprimates. Such germline genes may be isolated by various methodsconventional in the art, including, but not limited to, by hybridizationscreening of genomic libraries in bacteriophage λ or cosmid libraries,or by PCR amplification of genomic sequences using primers derived fromthe sequences shown in FIG. 12. Human genomic libraries are publiclyavailable or may be constructed de novo from human DNA.

Genomic clones of NF-AT_(c), particularly of the murine cognate NF-ATgene, may be used to construct homologous targeting constructs forgenerating cells and transgenic nonhuman animals having at least onefunctionally disrupted NF-AT_(c) allele, preferably homozygous forknocked out NF-AT_(c) alleles. Guidance for construction of homologoustargeting constructs may be found in the art, including: Rahemtulla etal. (1991) Nature 353: 180; Jasin et al. (1990) Genes Devel. 4: 157; Kohet al. (1992) Science 256:1210; Molina et al. (1992) Nature 357:161;Grusby et al. (1991) Science 253:1417; Bradley et al. (1992)Bio/Technology 10: 534, incorporated herein by reference). Homologoustargeting can be used to generate so-called “knockout” mice, which areheterozygous or homozygous for an inactivated NF-AT_(c) allele. Suchmice may be sold commercially as research animals for investigation ofimmune system development, neoplasia, T cell activation, signaltransduction, drug sreening, and other uses.

Chimeric targeted mice are derived according to Hogan, et al.,Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring HarborLaboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed., IRL Press, Washington, D.C.,(1987) which are incorporated herein by reference. Embryonic stem cellsare manipulated according to published procedures (Teratocarcinomas andEmbryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRLPress, Washington, D.C. (1987); Zjilstra et al. (1989) Nature 342:435;and Schwartzberg et al. (1989) Science 246: 799, each of which isincorporated herein by reference).

Additionally, a NF-AT_(c) cDNA or genomic gene copy may be used toconstruct transgenes for expressing NF-AT_(c) polypeptides at highlevels and/or under the transcriptional control of transcription controlsequences which do not naturally occur adjacent to the NF-AT_(c) gene.For example but not limitation, a constitutive promoter (e.g., a HSV-tkor pgk promoter) or a cell-lineage specific transcriptional regulatorysequence (e.g., a CD4 or CD8 gene promoter/enhancer) may be operablylinked to a NF-AT_(c)-encoding polynucleotide sequence to form atransgene (typically in combination with a selectable marker such as aneo gene expression cassette). Such transgenes can be introduced intocells (e.g., ES cells, hematopoietic stem cells) and transgenic cellsand transgenic nonhuman animals may be obtained according toconventional methods. Transgenic cells and/or transgenic nonhumananimals may be used to screen for antineoplastic agents and/or to screenfor potential immunomodulatory agents, as overexpression of NF-AT_(c) orinappropriate expression of NF-AT_(c) may result in a hyperimmune stateor enhance graft rejection reactions.

4. Antisense Polynucleotides

Additional embodiments directed to modulation of T cell activationinclude methods that employ specific antisense polynucleotidescomplementary to all or part of the sequences shown in FIG. 12. Suchcomplementary antisense polynucleotides may include nucleotidesubstitutions, additions, deletions, or transpositions, so long asspecific hybridization to the relevant target sequence corresponding toFIG. 12 is retained as a functional property of the polynucleotide.Complementary antisense polynucleotides include soluble antisense RNA orDNA oligonucleotides which can hybridize specifically to NF-AT_(c) mRNAspecies and prevent transcription of the mRNA species and/or translationof the encoded polypeptide (Ching et al. (1989) Proc. Natl. Acad. Sci.U.S.A. 86: 10006; Broder et al. (1990) Ann. Int. Med. 113: 604; Loreauet al. (1990) FEBS Letters 274: 53; Holcenberg et al., WO91/11535; U.S.Ser. No. 07/530,165; WO91/09865; WO91/04753; WO90/13641; and EP 386563,each of which is incorporated herein by reference). The antisensepolynucleotides therefore inhibit production of NF-AT_(c) polypeptides.Since NF-AT_(c) protein expression is associated with T lymphocyteactivation, antisense polynucleotides that prevent transcription and/ortranslation of mRNA corresponding to NF-AT_(c) polypeptides may inhibitT cell activation and/or reverse the activated phenotype of T cells.Compositions containing a therapeutically effective dosage of NF-AT_(c)antisense polynucleotides may be administered for treatment of immunediseases, including lymphocytic leukemias, and for inhibition oftransplant rejection reactions, if desired. Antisense polynucleotides ofvarious lengths may be produced, although such antisense polynucleotidestypically comprise a sequence of about at least 25 consecutivenucleotides which are substantially identical to a naturally-occurringNF-AT_(c) polynucleotide sequence, and typically which are identical toa sequence shown in FIG. 12.

Antisense polynucleotides may be produced from a heterologous expressioncassette in a transfectant cell or transgenic cell, such as a transgenicpluripotent hematopoietic stem cell used to reconstitute all or part ofthe hematopoietic stem cell population of an individual. Alternatively,the antisense polynucleotides may comprise soluble oligonucleotides thatare administered to the external milieu, either in the culture medium invitro or in the circulatory system or interstitial fluid in vivo.Soluble antisense polynucleotides present in the external milieu havebeen shown to gain access to the cytoplasm and inhibit translation ofspecific mRNA species. In some embodiments the antisense polynucleotidescomprise methylphosphonate moieties. For general methods relating toantisense polynucleotides, see Antisense RNA and DNA, (1988), D. A.Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

5. NF-AT_(c) Polypeptides

The nucleotide and amino acid sequences shown in FIG. 12 enable those ofskill in the art to produce polypeptides corresponding to all or part ofthe full-length human NF-AT_(c) polypeptide sequence. Such polypeptidesmay be produced in prokaryotic or eukaryotic host cells by expression ofpolynucleotides encoding NF-AT_(c), or fragments and analogs thereof.Alternatively, such polypeptides may be synthesized by chemical methodsor produced by in vitro translation systems using a polynucleotidetemplate to direct translation. Methods for expression of heterologousproteins in recombinant hosts, chemical synthesis of polypeptides, andin vitro translation are well known in the art and are described furtherin Maniatis et al., Molecular Cloning: A Laboratory Manual (1989), 2ndEd., Cold Spring Harbor, N.Y. and Berger and Kimmel, Methods inEnzymology, Volume 152, Guide to Molecular Cloning Techniques (1987),Academic Press, Inc., San Diego, Calif.

Fragments or analogs of NF-AT_(c) may be prepared by those of skill inthe art. Preferred amino- and carboxy-termini of fragments or analogs ofNF-AT_(c) occur near boundaries of functional domains. For example, butnot for limitation, such functional domains include: (1) domainsconferring the property of binding to other NF-AT components (e.g.,AP-1), (2) domains conferring the property of nuclear localization instimulated T lymphocytes, and (3) domains conferring the property ofenhancing activation of T cells when expressed at sufficient levels insuch cells. Additionally, such functional domains might include: (1)domains conferring the property of binding to RNA polymerase species,(2) domains having the capacity to directly alter local chromatinstructure, which may comprise catalytic activities (e.g.,topoisomerases, endonucleases) and/or which may comprise structuralfeatures (e.g., zinc fingers, histone-binding moieties), and (3) domainswhich may interact with accessory proteins and/or transcription factors.

One method by which structural and functional domains may be identifiedis by comparison of the nucleotide and/or amino acid sequence data shownin FIG. 12 to public or proprietary sequence databases. Preferably,computerized comparison methods are used to identify sequence motifs orpredicted protein conformation domains that occur in other proteins ofknown structure and/or function, such as the zinc fingers. For example,the NAD-binding domains of dehydrogenases, particularly lactatedehydrogenase and malate dehydrogenase, are similar in conformation andhave amino acid sequences that are detectably homologous (Proteins,Structures and Molecular Principles, (1984) Creighton (ed.), W. H.Freeman and Company, New York, which is incorporated herein byreference). Further, a method to identify protein sequences that foldinto a known three-dimensional structure are known (Bowie et al. (1991)Science 253: 164). Thus, the foregoing examples demonstrate that thoseof skill in the art can recognize sequence motifs and structuralconformations that may be used to define structural and functionaldomains in the NF-AT_(c) sequences of the invention. One example of adomain is the rel similarity region from amino acid 418 to amino acid710 of the NF-AT_(c) polypeptide sequence of FIG. 12.

Additionally, computerized comparison of sequences shown in FIG. 12 toexisting sequence databases can identify sequence motifs and structuralconformations found in other proteins or coding sequences that indicatesimilar domains of the NF-AT_(c) protein. For example but not forlimitation, the programs GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package (Genetics Computer Group, 575Science Dr., Madison, Wis.) can be used to identify sequences indatabases, such as GenBank/EMBL, that have regions of homology with aNF-AT_(c) sequences. Such homologous regions are candidate structural orfunctional domains. Alternatively, other algorithms are provided foridentifying such domains from sequence data. Further, neural networkmethods, whether implemented in hardware or software, may be used to:(1) identify related protein sequences and nucleotide sequences, and (2)define structural or functional domains in NF-AT_(c) polypeptides(Brunak et al. (1991) J. Mol. Biol. 220: 49, which is incorporatedherein by reference). For example, the 13-residue repeat motifs-SPRASVTEESWLG-(SEQ ID NO: 23) and -SPRVSVTDDSWLG-(SEQ ID NO: 24) areexamples of structurally related domains.

Fragments or analogs comprising substantially one or more functionaldomain may be fused to heterologous polypeptide sequences, wherein theresultant fusion protein exhibits the functional property(ies) conferredby the NF-AT_(c) fragment. Alternatively, NF-AT_(c) polypeptides whereinone or more functional domain have been deleted will exhibit a loss ofthe property normally conferred by the missing fragment.

By way of example and not limitation, the domain conferring the propertyof nuclear localization and/or interaction with AP-1 may be fused toβ-galactosidase to produce a fusion protein that is localized to thenucleus and which can enzymatically convert a chromogenic substrate to achromophore.

Although one class of preferred embodiments are fragments having amino-and/or carboxy-termini corresponding to amino acid positions nearfunctional domains borders, alternative NF-AT_(c) fragments may beprepared. The choice of the amino- and carboxy-termini of such fragmentsrests with the discretion of the practitioner and will be made based onexperimental considerations such as ease of construction, stability toproteolysis, thermal stability, immunological reactivity, amino- orcarboxyl-terminal residue modification, or other considerations.

In addition to fragments, analogs of NF-AT_(c) can be made. Such analogsmay include one or more deletions or additions of amino acid sequence,either at the amino- or carboxy-termini, or internally, or both; analogsmay further include sequence transpositions. Analogs may also compriseamino acid substitutions, preferably conservative substitutions.Additionally, analogs may include heterologous sequences generallylinked at the amino- or carboxy-terminus, wherein the heterologoussequence(s) confer a functional property to the resultant analog whichis not indigenous to the native NF-AT_(c) protein. However, NF-AT_(c)analogs must comprise a segment of 25 amino acids that has substantialsimilarity to a portion of the amino acid sequence shown in FIG. 12,respectively, and which has at least one of the requisite functionalproperties enumerated in the Definitions (supra). Preferred amino acidsubstitutions are those which: (1) reduce susceptibility to proteolysis,(2) reduce susceptibility to oxidation, (3) alter post-translationalmodification of the analog, possibly including phosphorylation, and (4)confer or modify other physicochemical or functional properties of suchanalogs, possibly including interaction with calcineurin orphophorylation or dephosphorylation thereby. NF-AT_(c) analogs includevarious muteins of a NF-AT_(c) sequence other than thenaturally-occurring peptide sequence. For example, single or multipleamino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally-occurring NF-AT_(c) sequence(preferably in the portion of the polypeptide outside the functionaldomains).

Conservative amino acid substitution is a substitution of an amino acidby a replacement amino acid which has similar characteristics (e.g.,those with acidic properties: Asp and Glu). A conservative (orsynonymous) amino acid substitution should not substantially change thestructural characteristics of the parent sequence (e.g., a replacementamino acid should not tend to break a helix that occurs in the parentsequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles, (1984) Creighton (ed.), W.H.Freeman and Company, New York; Introduction to Protein Structure,(1991), C. Branden and J. Tooze, Garland Publishing, New York, N.Y.; andThornton et al. (1991) Nature 354: 105; which are incorporated herein byreference).

Native NF-AT_(c) proteins, fragments thereof, or analogs thereof can beused as reagents in DNA binding assays and/or in vitro transcriptionassays for identifying agents that interfere with NF-AT function, saidagents are thereby identified as candidate drugs which may be used, forexample, to block T cell activation or treat T cell lymphocyticleukemias. Typically, in vitro DNA binding assays that measure bindingof NF-AT to DNA employ double-stranded DNA that contains an array of oneor more NF-AT recognition sites (as defined by specific footprinting ofnative NF-AT protein). The DNA is typically linked to a solid substrateby any of various means known to those of skill in the art; such linkagemay be noncovalent (e.g., binding to a highly charged surface such asNylon 66) or may be by covalent bonding (e.g., typically by chemicallinkage involving a nitrogen position in a nucleotide base, such asdiazotization). NF-AT_(c) polypeptides are typically labeled byincorporation of a radiolabeled amino acid. The labeled NF-AT_(c)polypeptide, usually reconstituted with an NF-AT nuclear component(e.g., AP-1 activity) to form an NF-AT complex, is contacted with theimmobilized DNA under aqueous conditions that permit specific binding incontrol binding reactions with a binding affinity of about 1×10⁶ M⁻¹ orgreater (e.g., 10-250 mM NaCl or KCl and 5-100 mM Tris HCl pH 5-9,usually pH 6-8), generally including Zn⁺² and/or Mn⁺² and/or Mg⁺² in thenanomolar to micromolar range (1 nM to 999 μM). Specificity of bindingis typically established by adding unlabeled competitor at variousconcentrations selected at the discretion of the practitioner. Examplesof unlabeled protein competitors include, but are not limited to, thefollowing: unlabeled NF-AT_(c) polypeptide, bovine serum albumin, andnuclear protein extracts. Binding reactions wherein one or more agentsare added are performed in parallel with a control binding reaction thatdoes not include an agent. Agents which inhibit the specific binding ofNF-AT_(c) polypeptides to DNA, as compared to a control reaction, areidentified as candidate immunomodulatory drugs. Also, agents whichprevent transcriptional modulation by NF-AT in vitro are therebyidentified as candidate immunomodulatory drugs.

In addition to NF-AT_(c) polypeptides consisting only ofnaturally-occuring amino acids, NF-AT_(c) peptidomimetics are alsoprovided. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. These types of non-peptide compound are termed“peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. DrugRes. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al.(1987) J. Med. Chem 30: 1229, which are incorporated herein byreference) and are usually developed with the aid of computerizedmolecular modeling. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to a paradigm polypeptide (i.e., a polypeptide thathas a biological or pharmacological activity), such as human NF-AT_(c),but have one or more peptide linkages optionally replaced by a linkageselected from the group consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—,—CH═CH— (cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methodsknown in the art and further described in the following references:Spatola, A. F. in “Chemistry and Biochemistry of Amino Acids, Peptides,and Proteins,” B. Weinstein, eds., Marcel Dekker, New York, p. 267(1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3,“Peptide Backbone Modifications” (general review); Morley, J. S., TrendsPharm Sci (1980) pp. 463-468 (general review); Hudson, D. et al., Int JPept Prot Res (1979) 14:177-185 (—CH₂NH—, CH₂CH₂—); Spatola, A. F. etal., Life Sci (1986) 38:1243-1249 (—CH₂—S); Hann, M. M., J Chem SocPerkin Trans I (1982) 307-314 (—CH—CH—, cis and trans); Almquist, R. G.et al., J Med Chem (1980) 23:1392-1398 (—COCH₂—); Jennings-White, C. etal., Tetrahedron Lett (1982) 23:2533 (—COCH₂—); Szelke, M. et al.,European Appln. EP 45665 (1982) CA: 97:39405 (1982) (—CH(OH)CH₂—);Holladay, M. W. et al., Tetrahedron Lett (1983) 24:4401-4404(—C(OH)CH₂—); and Hruby, V. J., Life Sci (1982) 31:189-199 (—CH₂—S—);each of which is incorporated herein by reference. A particularlypreferred non-peptide linkage is —CH₂NH—. Such peptide mimetics may havesignificant advantages over polypeptide embodiments, including, forexample: more economical production, greater chemical stability,enhanced pharmacological properties (half-life, absorption, potency,efficacy, etc.), altered specificity (e.g., a broad-spectrum ofbiological activities), reduced antigenicity, and others. Labeling ofpeptidomimetics usually involves covalent attachment of one or morelabels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) (e.g., immunoglobulinsuperfamily molecules) to which the peptidomimetic binds to produce thetherapeutic effect. Derivitization (e.g., labelling) of peptidomimeticsshould not substantially interfere with the desired biological orpharmacological activity of the peptidomimetic. Peptidomimetics ofNF-AT_(c) may be used as competitive or noncompetitive agonists orantagonists of NF-AT_(c) function. For example, a NF-AT_(c)peptidomimetic administered to a stimulated T cell containing NF-AT_(c)and may compete with the naturally-occurring NF-AT_(c) and reduce NF-ATactivity. Alternatively, an NF-AT_(c) peptidomimetic administerd to a Tcell lacking NF-AT_(c) may induce T cell activation or the like.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides (including cyclized peptides) comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch (1992) Ann. Rev. Biochem. 61: 387, incorporated herein byreference); for example, by adding internal cysteine residues capable offorming intramolecular disulfide bridges which cyclize the peptide.

The amino acid sequences of NF-AT_(c) polypeptides identified hereinwill enable those of skill in the art to produce polypeptidescorresponding to NF-AT_(c) peptide sequences and sequence variantsthereof. Such polypeptides may be produced in prokaryotic or eukaryotichost cells by expression of polynucleotides encoding a NF-AT_(c) peptidesequence, frequently as part of a larger polypeptide. Alternatively,such peptides may be synthesized by chemical methods. Methods forexpression of heterologous proteins in recombinant hosts, chemicalsynthesis of polypeptides, and in vitro translation are well known inthe art and are described further in Maniatis et al., Molecular Cloning:A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Bergerand Kimmel, Methods in Enzymology, Volume 152, Guide to MolecularCloning Techniques (1987), Academic Press, Inc., San Diego, Calif.;Merrifield, J. (1969) J. Am. Chem. Soc. 91: 501; Chaiken I. M. (1981)CRC Crit. Rev. Biochem. 11: 255; Kaiser et al.(1989) Science 243: 187;Merrifield, B. (1986) Science 232: 342; Kent, S. B. H. (1988) Ann. Rev.Biochem. 57: 957; and Offord, R. E. (1980) Semisynthetic Proteins, WileyPublishing, which are incorporated herein by reference).

Recombinant NF-AT polypeptides can be produced as follows.

The nucleic acid sequences of the present invention capable ofultimately expressing the desired NF-AT_(c) polypeptides can be formedfrom a variety of different polynucleotides (genomic or cDNA, RNA,synthetic oligonucleotides, etc.) as well as by a variety of differenttechniques.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to (i.e., positioned to ensurethe functioning of) an expression control sequence. These expressionvectors are typically replicable in the host organisms either asepisomes or as an integral part of the host chromosomal DNA. Commonly,expression vectors will contain selection markers, e.g., tetracyclineresistance or hygromycin resistance, to permit detection and/orselection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

E. coli is one prokaryotic host useful particularly for cloning the DNAsequences of the present invention. Other microbial hosts suitable foruse include bacilli, such as Bacillus subtilis, and otherEnterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, may also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(see, Winnacker, “From Genes to Clones,” VCH Publishers, N.Y., N.Y.(1987), which is incorporated herein by reference). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting intact human proteins have been developed in the art, andinclude the CHO cell lines, various COS cell lines, HeLa cells, myelomacell lines, Jurkat cells, etc. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter, an enhancer (Queen et al. (1986 Immunol. Rev. 89: 49, whichis incorporated herein by reference), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papillomavirus, and thelike. The vectors containing the DNA segments of interest (e.g.,polypeptides encoding a NF-AT_(c) polypeptide) can be transferred intothe host cell by well-known methods, which vary depending on the type ofcellular host. For example, CaCl transfection is commonly utilized forprokaryotic cells, whereas CaPO₄ treatment or electroporation may beused for other cellular hosts. (See, generally, Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,(1982), which is incorporated herein by reference). Usually, vectors areepisomes and are maintained extrachromosomally.

Expression of recombinant NF-AT_(c) protein in cells, particularly cellsof the lymphopoietic lineage, may be used to identify and isolate genesthat are transcriptionally modulated, either positively or negatively,by the presence of NF-AT_(c) protein. Such genes are typically initiallyidentified as cDNA clones isolated from subtractive cDNA libraries,wherein RNA isolated from cells expressing recombinant NF-AT and RNAisolated from control cells (i.e., not expressing recombinant NF-ATc)are used to generate the subtractive libraries and screening probes. Insuch a manner, NF-AT_(c)-dependent genes may be isolated. NFAT-dependentgenes (or their regulatory sequences operably linked to a reporter gene)may be used as a component of an in vitro transcription assay employinga NF-AT_(c) polypeptide as a necessary component for efficienttranscription; such transcription assays may be used to screen foragents which inhibit NF-AT_(c)-dependent gene transcription and arethereby identified as candidate immunomodulatory agents.

6. Production and Applications of α-NF-AT_(c) Antibodies

NF-AT_(c) and NF-ATn proteins, fragments thereof, or analogs thereof,may be used to immunize an animal for the production of specificantibodies. These antibodies may comprise a polyclonal antiserum or maycomprise a monoclonal antibody produced by hybridoma cells. For generalmethods to prepare antibodies, see Antibodies: A Laboratory Manual,(1988) E. Harlow and D. Lane, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., which is incorporated herein by reference.

For example but not for limitation, a recombinantly produced fragment ofhuman NF-AT_(c) or a purified NF-ATc protein can be injected into a rator a mouse along with an adjuvant following immunization protocols knownto those of skill in the art so as to generate an immune response.Typically, approximately at least 1-50 μg of a NF-AT_(c) fragment oranalog is used for the initial immunization, depending upon the lengthof the polypeptide. Alternatively or in combination with a recombinantlyproduced NF-AT_(c) polypeptide, a chemically synthesized peptide havinga NF-AT_(c) sequence (e.g., peptides exemplified in Table II, infra) maybe used as an immunogen to raise antibodies which bind a NF-AT_(c)protein, such as the native human NF-AT_(c) polypeptide having thesequence shown essentially in FIG. 12 or the native human NF-AT_(c)polypeptide isoform. Immunoglobulins which bind the recombinant fragmentwith a binding affinity of at least 1×10⁷ M⁻¹ can be harvested from theimmunized animal as an antiserum, and may be further purified byimmunoaffinity chromatography or other means. Additionally, spleen cellsare harvested from the immunized animal (typically rat or mouse) andfused to myeloma cells to produce a bank of antibody-secreting hybridomacells. The bank of hybridomas can be screened for clones that secreteimmunoglobulins which bind the recombinantly produced NF-AT_(c)polypeptide (or chemically synthesized NF-AT_(c) polypeptide) with anaffinity of at least 1×10⁶ M⁻¹. Animals other than mice and rats may beused to raise antibodies; for example, goats, rabbits, sheep, andchickens may also be employed to raise antibodies reactive with aNF-AT_(c) protein. Transgenic mice having the capacity to producesubstantially human antibodies also may be immunized and used for asource of α-NF-AT_(c) antiserum and/or for making monoclonal-secretinghybridomas.

Bacteriophage antibody display libraries may also be screened forbinding to a NF-AT_(c) polypeptide, such as a fill-length humanNF-AT_(c) protein, a NF-AT_(c) fragment (e.g., a peptide having asequence shown in Table II, infra), or a fusion protein comprising aNF-AT_(c) polypeptide sequence of at least 14 contiguous amino acids asshown in FIG. 12 or a polypeptide sequence of Table II (infra).Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313, incorporated herein by reference). Typically, a bacteriophageantibody display library is screened with a NF-AT_(c) polypeptide thatis immobilized (e.g., by covalent linkage to a chromatography resin toenrich for reactive phage by affinity chromatography) and/or labeled(e.g., to screen plaque or colony lifts).

NF-AT_(c) polypeptides which are useful as immunogens, for diagnosticdetection of α-NF-AT_(c) antibodies in a sample, for diagnosic detectionand quantitation of NF-AT_(c) protein in a sample (e.g., by standardizedcompetitive ELISA), or for screening a bacteriophage antibody displaylibrary, are suitably obtained in substantially pure form, that is,typically about 50 percent (w/w) or more purity, substantially free ofinterfering proteins and contaminants. Preferably, these polypeptidesare isolated or synthesized in a purity of at least 80 percent (w/w)and, more preferably, in at least about 95 percent (w/w) purity, beingsubstantially free of other proteins of humans, mice, or othercontaminants. Preferred immunogens comprise at least one NF-AT_(c)polypeptide sequence shown in Table II, either as a discrete peptide oras part of a fusion polypeptide (e.g., with a β-galactosidase orglutathione S-transferase sequence). NF-AT_(c) immunogens comprise atleast one, typically several of such immunogenic epitopes.

For some applications of these antibodies, such as identifyingimmunocrossreactive proteins, the desired antiserum or monoclonalantibody(ies) is/are not monospecific. In these instances, it may bepreferable to use a synthetic or recombinant fragment of NF-AT_(c) as anantigen rather than using the entire native protein. More specifically,where the object is to identify immunocrossreactive polypeptides thatcomprise a particular structural moiety, such as a DNA-binding domain,it is preferable to use as an antigen a fragment corresponding to partor all of a commensurate structural domain in the NF-AT_(c) protein.Production of recombinant or synthetic fragments having such definedamino- and carboxy-termini is provided by the NF-AT_(c) sequences shownin FIG. 12, may be accomplished by proteolytic digestion of a purifiedsubmit or produced recombinantly.

If an antiserum is raised to a NF-AT_(c) fusion polypeptide, such as afusion protein comprising a NF-AT_(c) immunogenic epitope fused toβ-galactosidase or glutathione S-transferase, the antiserum ispreferably preadsorbed with the non-NF-AT_(c) fusion partner (e.g,β-galactosidase or glutathione S-transferase) to deplete the antiserumof antibodies that react (i.e., specifically bind to) the non-NF-AT_(c)portion of the fusion protein that serves as the immunogen. Monoclonalor polyclonal antibodies which bind to the human and/or murine NF-AT_(c)protein can be used to detect the presence of human or murine NF-AT_(c)polypeptides in a sample, such as a Western blot of denatured protein(e.g., a nitrocellulose blot of an SDS-PAGE) obtained from a lymphocytesample of a patient. Preferably quantitative detection is performed,such as by denistometric scanning and signal integration of a Westernblot. The monoclonal or polyclonal antibodies will bind to the denaturedNF-AT_(c) epitopes and may be identified visually or by other opticalmeans with a labeled second antibody or labeled Staphylococcus aureusprotein A by methods known in the art. Frequently, denatured NF-AT_(c)will be used as the target antigen so that more epitopes may beavailable for binding.

TABLE II Selected Human NF-AT_(c) Antigen Peptides -NAIFLTVSREHERVGC-;(SEQ ID NO: 25) -LHGYLENEPLMLQLFIGT-; (SEQ ID NO: 26)-PSTSPRASVTEESWLG-; (SEQ ID NO: 27) -GPAPRAGGTMKSAEEEHYG-; (SEQ ID NO:28) -ASAGGHPIVQ-; (SEQ ID NO: 29) -NTRVRLVFRV-; (SEQ ID NO: 30)-AKTDRDLCKPNSLVVEIPPFRN-; (SEQ ID NO: 31) -EVQPKSHHRAHYETEGSR-; (SEQ IDNO: 32) -SPRVSVTDDSWLGNT-; (SEQ ID NO: 33) -SHHRAHYETEGSRGAV-; (SEQ IDNO: 34) -LRNSDIELRKGETDIGR-; and (SEQ ID NO: 35) -TLSLQVASNPIEC-. (SEQID NO: 36)

Such NF-AT_(c) sequences as shown in Tables II may be used as animmunogenic peptide directly (e.g., to screen bacteriophage antibodydisplay libraries or to immunize a rabbit), or may be conjugated to acarrier macromolecule (e.g., BSA) or may compose part of a fusionprotein to be used as an immunogen. A preferred NF-AT_(c) polypeptidecomprises the following amino acids sequences:

-NAIFLTVSREHERVGC-; (SEQ ID NO: 25) -PSTSPRASVTEESWLG-; (SEQ ID NO: 27)-SPRVSVTDDSWLGNT-; and (SEQ ID NO: 33) -SHHRAHYETEGSRGAV-; (SEQ ID NO:34)and may comprise other intervening and/or terminal sequences; generallysuch polypeptides are less than 1000 amino acids in length, more usuallyless than about 500 amino acids in length; often spacer peptidesequences or terminal peptide sequences, if present, correspond tonaturally occurring polypeptide sequences, generally mammalianpolypeptide sequences. One application of the preferred NF-AT_(c)polypeptide just recited is as a commercial immunogen to raiseα-NF-AT_(c) antibodies in a suitable animal and/or as a commercialimmunodiagnostic reagent for quantitative ELISA (e.g., competitiveELISA) or competitive RIA in conjunction with the anti-NF-AT_(c)antibodies provided by the invention, such as for calibration ofstandardization of such immunoassays for staging or diagnosis ofNF-AT_(c)-expressing lymphocytic leukemias in humans or cell typing oridentification of T cells (such as activated T cells and/or activatableT cells). The preferred NF-AT_(c) polypeptide just recited will findmany other uses in addition to serving as an immunogen or immunologicalreagent. One or more of the above-listed sequences may be incorporatedinto a fusion protein with a fusion partner such as human serum albumin,GST, etc. For such fusion proteins in excess of 1000 amino acids,deletions in the fusion partner (albumin) moiety may be made to bringthe size to about 1000 amino acids or less, if desired.

In some embodiments, it will be desirable to employ a polyvalentNF-AT_(c) antigen, comprising at least two NF-AT_(c) immunogenicepitopes in covalent linkage, usually in peptide linkage. Suchpolyvalent NF-AT_(c) antigens typically comprise multiple NF-AT_(c)antigenic peptides from the same species (e.g., human or mouse), but maycomprise a mix of antigenic peptides from NF-AT_(c) proteins ofdifferent species (i.e., an interspecies NF-AT_(c) polyvalent antigen).Frequently, the spatial order of the antigenic peptide sequences in theprimary amino acid sequence of a polyvalent antigen occurs in the sameorientation as in the naturally occurring NF-AT_(c) protein (i.e., afirst antigenic peptide sequence that is amino-terminal to a secondantigenic peptide sequence in a naturally occurring NF-AT_(c) proteinwill be amino-terminal to said second antigenic peptide sequence in apolyvalent antigen. Frequently, spacer peptide sequences will be used tolink antigenic peptide sequences in a polyvalent antigen, such spacerpeptide sequences may be predetermined, random, or psuedorandomsequences. Spacer peptide sequences may correspond to sequences known tobe non-immunogenic to the animal which is to be immunized with thepolyvalent antigen, such as a sequence to which the animal has beentolerized. Although many examples of such polyvalent antigens may begiven, the following embodiment is provided for illustration and notlimitation:

-NAIFLTVSREHERVGC-(aa1) (SEQ ID NO: 25) -AKTDRDLCKPNSLVVEIPPFRN-(aa2)(SEQ ID NO: 31) -GILKLRNSDIELRKGETD- (SEQ ID NO: 37)

where (aa1) and (aa2) are peptide spacers of at least one amino acid andless than 1000 amino acids; aa1 is a peptide sequence selectedindependently from the aa2 peptide sequence; the length of aa1 (whichmay be composed of multiple different amino acids) is independent of thelength of aa2 (which may be composed of multiple different amino acids).

Immunogenic NF-AT_(c) peptides may be used to immunize an animal toraise anti-NF-AT_(c) antibodies and/or as a source of spleen cells formaking a hybridoma library from which to select hybridoma clones whichsecrete a monoclonal antibody which binds to a NF-AT_(c) protein with anaffinity of 1×10⁷ M⁻¹ or greater, preferably at least 1×10⁸ M⁻¹ to 1×10⁹M⁻¹. Such immunogenic NF-AT_(c) peptides can also be used to screenbacteriophage antibody display libraries directly.

One use of such antibodies is to screen cDNA expression libraries,preferably containing cDNA derived from human or murine mRNA fromvarious tissues, for identifying clones containing cDNA inserts whichencode structurally-related, immunocrossreactive proteins, that arecandidate novel transcription factors or chromatin proteins. Suchscreening of cDNA expression libraries is well known in the art, and isfurther described in Young et al., Proc. Natl. Acad. Sci. U.S.A.80:1194-1198 (1983), which is incorporated herein by reference, as wellas other published sources. Another use of such antibodies is toidentify and/or purify immunocrossreactive proteins that arestructurally or evolutionarily related to the native NF-AT_(c) proteinor to the corresponding NF-AT_(c) fragment (e.g., functional domain;DNA-binding domain) used to generate the antibody. It is believed thatsuch antibodies will find commercial use as such reagents for researchapplications, just as other antibodies (and biological reagents—such asrestriction enzymes and polymerases) are sold commercially.

Various other uses of such antibodies are to diagnose and/or stageleukemias or other immunological disease states, and for therapeuticapplication (e.g., as cationized antibodies or by targeted liposomaldelivery) to treat neoplasia, hyperimmune function, graft rejection, andthe like.

An example of an NF-ATc polypeptide is a polypeptide having thesequence:

MPSTSFPVPSKFPLGPAAAVFGRGETLGPAPRAG (SEQ ID NO: 38)GTMKSAEEEHYGYASSNVSPALPLPTAHSTLPAP CHNLQTSTPGIIPPADHPSGYGAALDGCPAGYFLSSGHTRPDGAPALESPRIEITSCLGLYHNNNQFF HDVEVEDVLPSSKRSPSTATLSLPSLEAYRDPSCLSPASSLSSRSCNSEASSYESNYSYPYASPQTSP WQSPCVSPKTTDPEEGFPRGLGACTLLGSPQHSPSTSPRASVTEESWLGARSSRPASPCNKRKYSLNG RQPPYSPHHSPTPSPHGSPRVSVTDDSWLGNTTQYTSSAIVAAINALTTDSSLDLGDGVPVKSRKTTL EQPPSVALKVEPVGEDLGSPPPPADFAPEDYSSFQHIRKGGFCDQYLAVPQHPYQWAKPKPLSPTSYM SPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASAGGHPIVQLHGYLENEPLMLQL FIGTADDRLLRPHAFYQVHRITGKTVSTTSHEAILSNTKVLEIPLLPENSMRAVIDCACILKLRNSDI ELRKGETDIGRKNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDSYPVVGGK KMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNSLVVEIPPFRNQRITSPVHVSGYVCN GKRKRSQYQRFTYLPANGNAIFLTVSREHERVGC FF7. Identification and Isolation of Proteins that Bind NF-AT_(c)

Proteins that bind to NF-AT_(c) NF-ATn, NF-ATc:NF-ATn and/or a NFAT-DNAcomplex are potentially important transcriptional regulatory proteins.Such proteins may be targets for novel immunomodulatory agents. Theseproteins are referred to herein as accessory proteins. Accessoryproteins may be isolated by various methods known in the art.

One preferred method of isolating accessory proteins is by contacting aNF-AT_(c) polypeptide to an antibody that binds the NF-AT_(c)polypeptide, and isolating resultant immune complexes. These immunecomplexes may contain accessory proteins bound to the NF-AT_(c)polypeptide. The accessory proteins may be identified and isolated bydenaturing the immune complexes with a denaturing agent and, preferably,a reducing agent. The denatured, and preferably reduced, proteins can beelectrophoresed on a polyacrylamide gel. Putative accessory proteins canbe identified on the polyacrylamide gel by one or more of various wellknown methods (e.g., Coomassie staining, Western blotting, silverstaining, etc.), and isolated by resection of a portion of thepolyacrylamide gel containing the relevant identified polypeptide andelution of the polypeptide from the gel portion.

A putative accessory protein may be identified as an accessory proteinby demonstration that the protein binds to NF-AT_(c) and/or a NFAT-DNAcomplex. Such binding may be shown in vitro by various means, including,but not limited to, binding assays employing a putative accessoryprotein that has been renatured subsequent to isolation by apolyacrylamide gel electrophoresis method. Alternatively, binding assaysemploying recombinant or chemically synthesized putative accessoryprotein may be used. For example, a putative accessory protein may beisolated and all or part of its amino acid sequence determined bychemical sequencing, such as Edman degradation. The amino acid sequenceinformation may be used to chemically synthesize the putative accessoryprotein. The amino acid sequence may also be used to produce arecombinant putative accessory protein by: (1) isolating a cDNA cloneencoding the putative accessory protein by screening a cDNA library withdegenerate oligonucleotide probes according to the amino acid sequencedata, (2) expressing the cDNA in a host cell, and (3) isolating theputative accessory protein. Alternatively, a polynucleotide encoding aNF-AT_(c) polypeptide may be constructed by oligonucleotide synthesis,placed in an expression vector, and expressed in a host cell.

Yeast two-hybrid systems may be used to screen a mammalian (typicallyhuman) cDNA expression library, wherein cDNA is fused to a GAL4 DNAbinding domain or activator domain, and a NF-AT_(c) polypeptide sequenceis fused to a GAL4 activator domain or DNA binding domain, respectively.Such a yeast two-hybrid system can screen for cDNAs encoding proteinswhich bind to NF-AT_(c) sequences. For example, a cDNA library can beproduced from mRNA from a human mature T cell line or other suitablecell type. Such a cDNA library cloned in a yeast two-hybrid expressionsystem (Chien et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 9578 orCell 72: 233) can be used to identify cDNAs which encode proteins thatinteract with NF-AT_(c) and thereby produce expression of theGAL4-dependent reporter gene. Polypeptides which interact with NF-AT_(c)can alos be identified by immunoprecipitation of NF-AT_(c) with antibodyand identification of co-precipitating species. Further, polypeptidesthat bind NF-AT_(c) can be identified by screening a peptide library(e.g., a bacteriophage peptide display library, a spatially definedVLSIPS peptide array, and the like) with a NF-AT_(c) polypeptide.Accessory proteins may also be identified by crosslinking in vivo withbifunctional crosslinking reagents (e.g., dimethylsuberimidate,glutaraldehyde, etc.) and subsequent isolation of crosslinked productsthat include a Lyar polypeptide. For a general discussion ofcross-linking, see Kunkel et al. (1981) Mol. Cell. Biochem. 34: 3, whichis incorporated herein by reference. Preferably, the bifunctionalcrosslinking reagent will produce crosslinks which may be reversed underspecific conditions after isolation of the crosslinked complex so as tofacilitate isolation of the accessory protein from the NF-AT_(c)polypeptide. Isolation of crosslinked complexes that include a NF-AT_(c)polypeptide is preferably accomplished by binding an antibody that bindsa NF-AT_(c) polypeptide with an affinity of at least 1×10⁷ M⁻¹ to apopulation of crosslinked complexes and recovering only those complexesthat bind to the antibody with an affinity of at least 1×10⁷ M⁻¹.Polypeptides that are crosslinked to a NF-AT_(c) polypeptide areidentified as accessory proteins.

Screening assays can be developed for identifying candidateimmunomodulatory agents as being agents which inhibit binding ofNF-AT_(c) to an accessory protein (e.g. AP-1) under suitable bindingconditions.

8. Methods for Identifying Compounds which Affect NF-AT Activity

These methods of screening may involve labeling NF-ATc, or correspondingpeptide with any of a myriad of suitable markers, including radiolabels(e.g., ¹²⁵I or ³²P), various fluorescent labels and enzymes, (e.g.,glutathione-S-transferase and β-galactosidase). If desired for basicbinding assays, one of the components may be immobilized by standardtechniques, with the non-immobilized component typically being labeled.

The screening assays of the present invention may utilize isolated orpurified forms of these assay components. This refers to nucleic acidsegments, polypeptides and the like of the present invention which havebeen separated from their native environment (e.g., a cytoplasmic ornuclear fraction of a cell, to at least about 10-50% purity. Asubstantially pure composition includes such compounds that areapproaching homogeneity, i.e., about 80-90% pure, preferably 95-99%pure.

While any of the standard pharmaceutical sources of therapeuticcandidate agents may be used, a preferred class of agents suitable foruse in the screening assays of the present invention are macrolides,particularly those exhibiting a twisted amide peptidyl prolyl bond. See,Schreiber, Science, 251, 283-287 (1991) and Banerji et al., Mol. andCell. Biol., 11, 4074-4087 (1991). These compounds are also preferablycapable of binding to and. blocking the cystolic receptors FKBP-12 andFKBP-13. See, Jin et al., Proc. Natl. Acad. Sci., U.S.A., 88, 6671-6681(1991).

Agent screening using the methods of the present invention can befollowed by biological testing to determine if the compound has thedesired activities in vitro and in vivo. The ultimate therapeutic agentmay be administered directly to the host to be treated. Therapeuticformulations may be administered in any conventional dosage formulation.While for the active ingredient may be administered alone, preferably,it is presented as a pharmaceutical formulation. Formulations compriseat least one active ingredient as defined above together with one ormore pharmaceutically acceptable carriers thereof. Each carrier must beboth pharmaceutically and physiologically acceptable in the sense ofbeing compatible with the other ingredients and not injurious to thepatient. Formulations include those suitable for oral, rectal, nasal, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration of a therapeutically effective dose. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy.

NF-AT_(c), NF-AT_(n) or fragments thereof produced by proteolyticcleavage or recombinantly, can be used as reagents in heterodimerizationassays for identifying agents that disrupt NF-AT complex formation, saidagents are thereby identified as candidate immunosuppressant drugs.Alternatively, these polypeptides can be used in in vitro assaysmeasuring binding of heterodimeric NF-AT to NF-AT recognition sequencesand/or with in vitro transcription assays which measure the ability ofNF-AT to enhance the rate of transcription of a sequence linked to atleast a minimal promoter and an NF-AT recognition sequence. Typically,in vitro assays that measure binding of NF-AT to DNA employdouble-stranded DNA that contains an array of one or more NF-ATrecognition sites. The DNA is typically linked to a solid substrate byany of various means known to those of skill in the art; such linkagemay be noncovalent (e.g., binding to a highly charged surface such asNylon 66) or may be by covalent bonding (e.g., typically by chemicallinkage involving a nitrogen position in a nucleotide base, such asdiazotization) NF-AT_(c) and/or NF-AT_(n) are typically labeled byincorporation of a radiolabeled amino acid. The labeled NF-AT protein iscontacted with the immobilized DNA under aqueous conditions that permitspecific binding in control binding reactions of 1×10⁶ M⁻¹ or greater(e.g., 20-150 mM NaCl and 5-100 mM Tris HCl pH 6-8). Specificity ofbinding is typically established by adding unlabeled competitor atvarious concentrations selected at the discretion of the practitioner.Examples of unlabeled protein competitors include, but are not limitedto, the following: unlabeled NF-AT_(c) polypeptide, unlabeled NF-AT_(n),polypeptide, bovine serum albumin, and nuclear protein extracts. Bindingreactions wherein one or more agents are added are performed in parallelwith a control binding reaction that does not include an agent. Agentswhich inhibit the specific binding of NF-AT protein to DNA, as comparedto a control reaction, are identified as candidate immunosuppressants.Also, agents which prevent in vitro heterodimer formation of NF-ATand/or prevent transcriptional enhancement by NF-AT in vitro are therebyidentified as candidate immunosuppressant drugs.

The screening assays of the present invention may utilize isolated,purified, or recombinant forms of these assay components. This refers,e.g., to purified polypeptides and the like of the present inventionwhich have been separated from their native environment (e.g., acytoplasmic or nuclear fraction of a cell), to at least about 10-50%purity. A substantially pure composition includes such agents that areapproaching homogeneity, i.e., about 80-90% pure, preferably 95-99%pure.

8.1. Methods Involving DNA Binding Assays

Candidate immunosuppressants can be identified by NF-AT DNA bindingassays. Some candidate immunosuppressants have the ability to inhibitthe binding of an assembled NF-AT complex, and, in some instances, ofindividual NF-AT polypeptides to DNA, particularly where the DNA isdouble-stranded and has at least one NF-AT recognition site sequence(e.g., as shown in Table I). Various means for detecting specificbinding between the NF-AT complex or NF-AT subunit polypeptide andtarget DNA can be used. Agents which inhibit specific binding of NF-ATcomplex or NF-AT subunit polypeptide to target DNA are identified ascandidate immunosuppressants.

Within the interleukin-2 enhancer (FIG. 2), there are two NF-AT sites, aproximal and distal NF-AT site. The sequence of these is shown in detailin FIG. 3. The essential residues judged by methylation interference areindicated by the lower case letters. Elimination of the NF-AT site fromthe IL-2 enhancer drastically reduces the ability of the enhancer tofunction. In addition, arrays of the binding site for the NF-AT proteinwill direct expression of linked sequences (e.g., reporter or toxingenes) to a specific biologic circumstance, notably the activated Tlymphocyte wherein transcriptionally active NF-AT complexes are formed.

A distinguishing feature of the NF-AT DNA binding site upstream of theIL-2 gene is its purine-rich binding site 5′-AAGAGGAAAAA-3′ (SEQ IDNO:53). DNA sequence comparisons of the promoter/enhancer regions ofseveral genes that respond to T cell activation signals has identifiedputative NF-AT protein binding sites. Such a comparison suggests thatNF-AT or a related family member may bind within the promoter/enhancerregions of other T cell activation dependent genes. Most of these genesare sensitive to immunosuppressants, such as FK506 and cyclosporin. Alist of putative NF-AT binding sites follows in Table III:

TABLE III Purine Position Core Sequences Position GeneGAAAGGAGGAAAAACTGTTT (−289 to −270) human IL-2 (SEQ ID NO: 54)CCAAAGAGGAAAATTTGTTT (−293 to −274) murine IL-2 (SEQ ID NO: 55)CAGAAGAGGAAAAATGAAGG (−143 to −124) human IL-2 (SEQ ID NO: 56)TCCAGGAGAAAAAATGCCTC (−143 to −124) human IL-4 (SEQ ID NO: 57)AAAACTTGIGAAAATACGTA (−71 to −52) human γ-IFN (SEQ ID NO: 58)TAAAGGAGAGAACACCAGCT (−270 to −251) HIV-LTR (SEQ ID NO: 59)GCAGGGTGGGAAAGGCCTTT (−241 to −222) murine GM-CSF (SEQ ID NO: 59)(Abbreviations: IL-2, interleukin 2; IL-4, interleukin 4; HIV-LTR, humanimmunodeficiency virus long terminal repeat; GM-CSF,granulocyte-macrophage colony stimulating factor.)

Other NF-AT specific nucleic acid binding sites, usually at least about10-150 nucleotides (which may be part of a much longer sequence)substantially homologous to these sequences, particularly the NF-AT DNAbinding site of the IL-2 enhancer. Ordinarily, such sequences conferNF-AT-dependent transcriptional enhancement on linked (i.e., within 1-75kb) promoters and are at least about 80% homologous to the NF-AT DNAbinding site, preferably in excess of 90% homologous or more, mostpreferably are identical.

NF-AT binding assays generally take one of two forms: immobilized targetDNA can be used to bind labeled NF-AT protein(s), or conversely,immobilized NF-AT protein(s) can be used to bind labeled target DNA. Ineach case, the labeled macromolecule (protein or DNA) is contacted withthe immobolized macromolecule (respectively, DNA or protein) underaqueous conditions that permit specific binding of the NF-AT protein(s)to the target DNA. Particular aqueous conditions may be selected by thepractitioner according to conventional methods, including methodsemployed in DNA-protein footprinting and/or in vitro nuclear run-ontranscription (Dunn et al. J. Biol. Chem. 263: 10878-10886 (1988), whichis incorporated herein by reference). However, preferable embodimentsutilize the following buffered aqueous conditions: 20-150 mM NaCl, 5-50mM Tris HCl, pH 5-8. It is appreciated by those in the art thatadditions, deletions, modifications (such as pH) and substitutions (suchas KCl substituting for NaCl or buffer substitution) may be made tothese basic conditions. Modifications can be made to the basic bindingreaction conditions so long as specific binding of NF-AT protein(s) totarget DNA occurs. Conditions that do not permit specific binding incontrol reactions (no agent included) are not suitable for use in DNAbinding assays.

In embodiments where target DNA is immobilized, preferablydouble-stranded DNA containing at least one NF-AT recognition sitesequence is bonded, either covalently or noncovalently, to a substrate.For example, but not for limitation, DNA can be covalently linked to adiazotized substrate, such as diazotized cellulose, particularlydiazophenylthioether cellulose and diazobenzyloxymethyl cellulose(Alwine et al. Proc. Natl. Acad. Sci. (U.S.A.) 74: 5350 (1977); Reiseret al. Biochem. Biophys. Res. Commun. 85: 1104 (1978); Stellwag andDahlberg, Nucleic Acids Res. 8: 299 (1980), which are incorporatedherein by reference).

Alternatively, DNA can be covalently linked to a substrate by partialultraviolet light-induced crosslinking to a Nylon 66 or nitrocellulosesubstrate (Church and Gilbert, Proc. Natl. Acad Sci. (U.S.A.) 81: 1991(1984), which is incorporated herein by reference). Also, for exampleand not for limitation, DNA can be noncovalently bound to a Nylon 66 orother highly charged anionic substrate (Berger and Kimmel, Methods inEnzymology, Volume 152, Guide to Molecular Cloning Techniques (1987),Academic Press, Inc., San Diego, Calif.). In some embodiments, it ispreferable to use a linker or spacer to reduce potential sterichindrance from the substrate. The immobilized DNA is contacted withlabeled NF-AT protein(s), such as NF-AT_(n), NFAT_(c), or fragments ofNF-AT_(n) and NF-AT_(c), or complexes thereof (including[NF-AT_(c):NF-AT_(n)] heterodimers).

Preferably, at least one NF-AT protein or NF-AT polypeptide species islabeled with a detectable marker. Suitable labeling includes, but is notlimited to, radiolabeling by incorporation of a radiolabeled amino acid(e.g., C¹⁴-labeled leucine, H³-labeled glycine, S³⁵,-labeledmethionine), radiolabeling by post-translational radioiodination withI¹²⁵ or I¹³¹ (e.g., Bolton-Hunter reaction and chloramine T), labelingby post-translational phosphorylation with P³² (e.g., phosphorylase andinorganic radiolabeled phosphate, calcineurin), fluorescent labeling byincorporation of a fluorescent label (e.g., fluorescein or rhodamine),or labeling by other conventional methods known in the art. Inembodiments where the target DNA is immobilized by linkage to asubstrate, at least one species of NF-AT polypeptide is labeled with adetectable marker.

In DNA binding assay embodiments where two or more species of NF-ATsubunit polypeptide are used concomitantly, for example a NF-AT_(n)polypeptide and a NF-AT_(c) polypeptide, at least one NF-AT subunitpolypeptide species is labeled. Additionally, in some embodiments wheremore than one NF-AT species are employed, it is preferred that differentlabels are used for each polypeptide, so that binding of individualand/or heterodimeric and/or multimeric NF-AT complexes to target DNA canbe distinguished. For example but not limitation, a NF-AT_(c)polypeptide may be labeled with fluorescein and a NF-AT_(n) polypeptidemay be labeled with a fluorescent marker that fluorescesces with eithera different excitation wavelength or emission wavelength, or both.Alternatively, double-label scintillation counting may be used, whereinone NF-AT subunit polypeptide is labeled with one isotope (e.g., H³) anda second NF-AT subunit polypeptide is labeled with a different isotope(e.g., C¹⁴) that can be distinguished by scintillation counting usingdiscrimination techniques.

Labeled NF-AT subunit polypeptides are contacted with immobilized DNAtarget under aqueous conditions as described infra. The time andtemperature of incubation of a binding reaction may be varied, so longas the selected conditions permit specific binding to occur in a controlreaction where no agent is present. Preferable embodiments employ areaction temperature of at least 20 degrees Centigrade, more preferably35 to 42 degrees Centigrade, and a time of incubation of at least 15seconds, although longer incubation periods are preferable so that, insome embodiments, a binding equilibrium is attained. Binding kineticsand the thermodynamic stability of bound NF-AT:DNA complexes determinethe latitude available for varying the time, temperature, salt, pH, andother reaction conditions. However, for any particular embodiment,desired binding reaction conditions can be calibrated readily by thepractitioner using conventional methods in the art, which may includebinding analysis using Scatchard analysis, Hill analysis, and othermethods (Proteins, Structures and Molecular Principles, (1984) Creighton(ed.), W.H. Freeman and Company, New York).

Specific binding of labeled NF-AT protein to immobilized DNA isdetermined by including unlabeled competitor protein(s) (e.g., albumin)and/or unlabeled competitor DNA or competitor oligonucleotides. After abinding reaction is completed, labeled NF-AT protein(s) that isspecifically bound to immobilized target DNA is detected. For exampleand not for limitation, after a suitable incubation period for binding,the aqueous phase containing non-immobilized protein and nucleic acid isremoved and the substrate containing the target DNA and any labeledprotein bound to the DNA is washed with a suitable buffer, optionallycontaining unlabeled blocking agent(s), and the wash buffer(s) removed.After washing, the amount of detectable label remaining specificallybound to the immobilized DNA is determined (e.g., by optical, enzymatic,autoradiographic, or other radiochemical methods).

In some embodiments, addition of unlabeled blocking agents that inhibitnon-specific binding are included. Examples of such blocking agentsinclude, but are not limited to, the following: calf thymus DNA, salmonsperm DNA, yeast RNA, mixed sequence (random or pseudorandom sequence)oligonucleotides of various lengths, bovine serum albumin, nonionicdetergents (NP-40, Tween, Triton X-100, etc.), nonfat dry milk proteins,Denhardt's reagent, polyvinylpyrrolidone, Ficoll, and other blockingagents. Practioners may, in their discretion, select blocking agents atsuitable concentrations to be included in DNA binding assays; howeverreaction conditions are selected so as to permit specific bindingbetween a NF-AT protein and target DNA in a control binding reaction.Blocking agents are included to inhibit nonspecific binding of labeledNF-AT protein to immobilized DNA and/or to inhibit nonspecific bindingof labeled DNA to immobilized NF-AT protein.

In embodiments where protein is immobilized, covalent or noncovalentlinkage to a substrate may be used. Covalent linkage chemistriesinclude, but are not limited to, well-characterized methods known in theart (Kadonaga and Tijan, Proc. Natl. Acad. Sci. (U.S.A.) 83: 5889-5893(1986), which is incorporated herein by reference). One example, not forlimitation, is covalent linkage to a substrate derivatized with cyanogenbromide (such as CNBr-derivatized Sepharose 4B). It may be desirable touse a spacer to reduce potential steric hindrance from the substrate.Noncovalent bonding of proteins to a substrate include, but are notlimited to, bonding of the protein to a charged surface and binding withspecific antibodies. DNA is typically labeled by incorporation of aradiolabeled nucleotide (H³, C¹⁴, S³⁵, p³²) or a biotinylated nucleotidethat can be detected by labeled avidin (e.g., avidin containing afluorescent marker or enzymatic activity).

NF-AT proteins may exhibit at least three levels of specific bindingproperty: (1) binding to DNA, (2) binding to double-stranded DNA, (3)binding to DNA containing at least one NF-AT recognition site sequence.Each level of binding specificity may be a potential target forcandidate immunosuppressants. The DNA assay systems described above maybe tailored to assay for each type of binding specificity, if desired.

8.2. Methods for Assaying Heterodimerization

Methods of screening for agents that reduce the binding of the NF-AT_(n)subunit to the NF-AT_(c) subunit, and more particularly that prevent thespecific heterodimerization of these two subunits, also can identifynovel candidate immunosuppressants. Heterodimerization assays involve invitro binding assays comprising NF-AT_(n) and NF-AT_(c) polypeptides(native, fragments, or analogs), wherein test agents can be added to thebinding reaction(s) and tested for their ability to inhibit heterodimerformation or reduce the affinity of binding. Agents which interfere withthe intermolecular binding between the NF-AT_(n) subunit (or fragmentthereof) and the NF-AT_(c) subunit (or fragment thereof) are therebyidentified as candidate immunosupressants.

These methods of screening may involve labeling NF-AT_(n), NF-AT_(c), orcorresponding fragments or analogs with any of a myriad of suitablemarkers, including radiolabels (e.g., ¹²⁵I or ³²P) various fluorescentlabels and enzymes, (e.g., glutathione-S-transferase, luciferase, andβ-galactosidase). If desired for basic binding assays, one of thecomponents may be immobilized by standard techniques. For example butnot for limitation, such immobilization may be effected by linkage to asolid support, such as a chromatographic matrix, or by binding to acharged surface, such as a plastic 96-well microtiter dish.

In one class of embodiments, parallel heterodimerization reactions areconducted, wherein one set of reactions serves as control and at leastone other set of reactions include various quantities of agents,mixtures of agents, or biological extracts, that are being tested forthe capacity to inhibit pairwise heterodimerization between a NF-AT_(c)polypeptide (native or fragment) and a NF-AT_(n) polypeptide (native orfragment). Agents that inhibit heterodimerization relative to thecontrol reaction(s) are thereby identified as candidateimmunosuppressants.

Preferred embodiments include heterodimerization assays which useNF-AT_(n) and NF-AT_(c) polypeptides which are produced by purificationfrom lymphocytes, particularly T lymphocytes (e.g., Jurkat cells).

8.3. Methods Involving In Vitro Transcription

Methods of screening for agents that inhibit in vitro transcription oftemplate polynucleotides which comprise at least one NF-AT recognitionsequence can also be used to identify candidate immunosuppressants. Invitro transcription reactions that are dependent on the presence offunctional (NF-AT_(n):NF-AT_(c)) heterodimer can serve as the basis forsuch screening assays. Such screening assays employ purified NF-AT, andNF-AT_(n) subunits, or fragments thereof, which retain the capacity toform functional [NF-AT_(n):NF-AT_(c)] heterodimers that can interactwith NF-AT recognition sequences and enhance in vitro transcription oftemplate polynucleotides comprising linked NF-AT recognition sequencesand at least a minimal promoter.

NF-AT-dependent in vitro transcription reactions are defined herein asreactions wherein the addition of an effective amount of NF-ATheterodimer produces a measurable increase in the amount oftranscription product(s) and/or increases the accuracy or frequency oftranscriptional initiation as compared to a parallel control reactionwhich does not contain NF-AT. Thus, NF-AT-dependent transcription isthat portion of the total transcription that is attributable to thepresence of NF-AT. Experimental conditions for in vitro transcriptionassays may be selected at the discretion of the practitioner accordingto methods known in the art, or may be done according to Flanagan andCrabtree, J. Biol. Chem. 267: 915 (1992), which is incorporated hereinby reference.

Agents which inhibit NF-AT-dependent transcription in such in vitrotranscription assays are thereby identified as candidateimmunosuppressants.

For example and not for limitation, one embodiment of such an in vitrotranscription assay employs a transcription template that is apolynucleotide comprising at least one NF-AT recognition site linked toa minimal promoter and some additional downstream transcribed sequences.The in vitro transcription reaction cocktail comprises the templatepolynucleotide, an NF-AT_(c) polypeptide species (native or fragment),an NF-AT_(n) polypeptide species (native or fragment), an RNA polymerasespecies, preferably human RNA polymerase II, ribonucleotides, and otherconstituents which are typically included in transcription reactioncocktails. See Heintz and Roeder, Genetic Engineering (1982) PlenumPress, New York. The reactions may be conducted as described in Flanaganand Crabtree, J. Biol. Chem. 267: 915 (1992). Where at least one of theribonucleotide species is radiolabeled, transcription products of thereaction are electrophoresed on a polyacrylamide gel and autoradiographyis performed to identify the size and relative amount(s) oftranscription product(s). Parallel in vitro transcription reactions areconducted, wherein one set of reactions serves as control and at leastone other set of reactions include various quantities of agents,mixtures of agents, or biological extracts that are being tested for thecapacity to inhibit (or enhance) in vitro transcription of the template.Agents that inhibit the in vitro transcription relative to the controlreaction(s) are thereby identified as candidate immunosuppressants.Agents which enhance transcription may be novel transcription factors oradditional protein factors that participate in NF-AT-mediatedtranscriptional enhancement.

One preferred embodiment of an in vitro transcription assay employs atranscription template comprising the nucleotide sequence which is the325 nucleotides immediately upstream from the transcriptional start siteof the human IL-2 gene.

Another preferred embodiment employs a transcription template thatcomprises a minimal promoter and a linked tandem array of three NF-ATrecognition sequences.

Additionally, preferred embodiments comprise NF-AT_(n), and NF-AT_(c)polypeptides that are produced by purification from lymphocytic cells.Biological activity of NF-AT subunit polypeptides can be modified byaltering post-translational modifications, such as phosphorylation. Invivo, Ca²⁺⁻ and calmodulin-dependent phosphatase activity, such ascalcineurin (Liu et al. Cell 66: 807-815 (1991); Friedman and WeissmanCell 66: 799-806 (1991), which are incorporated herein by reference),are involved in modulating biological activity of NF-AT. Therefore, insome embodiments, it is desirable to alter NF-AT polypeptides bypost-translational modifications, such as phosphorylation (e.g., with akinase) or dephosphorylation (e.g., with a phosphorylase).

For example but not for limitation, a NF-AT subunit can be partiallyphosphorylated by incubation with phosphorylase and inorganic phosphateunder conventional reaction conditions known in the art. Alternatively,a NF-AT subunit polypeptide can be partially dephosphorylated byincubation with calf intestinal alkaline phosphatase under conventionalreaction conditions. Incubation of NF-AT polypeptides or aggregatedNF-AT complex with calcineurin may also be employed. The degree ofphosphorylation or dephosphorylation that is achieved with suchenzymatic treatments may be determined at the discretion of thepractitioner by altering one or more of the following conditions: enzymeconcentration, substrate concentration, inorganic phosphateconcentration, temperature and duration of incubation, and otherreaction parameters known to those of skill in the art.

8.4 Methods for Identifying Compounds which Prevent NF-ATc NuclearTranslocation

Methods for screening compounds that prevent the NF-AT_(c) componentfrom translocating to the nucleus are preferably based on theobservation that immunosuppressants, such as FK506 and CsA, inhibitNF-AT_(c) from entering the nucleus of FK506 and CsA treated T cells.This inhibition may occur by modifying the NF-AT_(c) component so thatNF-AT_(c) is unable to engage in entry to the nucleus. Thus, an assaytypically involves a polypeptide comprising a peptide region ofNF-AT_(c) which becomes modified (e.g., by phophorylation) upon T cellactivation, wherein this polypeptide is used to screen compounds whichinhibit or enhance the modification of NF-AT_(c) all in accordance withstandard procedures, such as determining whether or not the modificationhas occurred by performing polyacrylamide gel electrophoresis on samplesobtained subsequent to T cell activation and identifying the relativemobility of NF-AT_(c), by immunoreactivity (e.g., Western blotting)and/or autoradiography (e.g., ³²P if the modification isphosphorylation).

Alternatively, the nuclear pore of the T cell may be altered to prevententry of NF-ATc into the nucleus. Such an assay involves analyzingtranslocation of NF-AT_(c), or a corresponding peptide into nuclei thathad been previously treated with compounds which alter the nuclear poreof the T cell so that NF-AT_(c) translocation through or associationwith the nuclear pore structure fails to occur.

8.5. Methods for Rational Drug Design

NF-AT_(c) polypeptides, especially those portions which form directcontacts in NF-AT complexes, can be used for rational drug design ofcandidate NFAT-modulating agents (e.g., antineoplastics andimmunomodulators). The substantially purified NF-AT_(c) and theidentification of NF-AT_(c) as a docking partner for AP-1 activities asprovided herein permits production of substantially pure NF-ATpolypeptide complexes and computational models which can be used forprotein X-ray crystallography or other structure analysis methods, suchas the DOCK program (Kuntz et al. (1982) J. Mol. Biol. 161: 269; Kuntz ID (1992) Science 257: 1078) and variants thereof. Potential therapeuticdrugs may be designed rationally on the basis of structural informationthus provided. In one embodiment, such drugs are designed to preventformation of a NF-AT_(c) polypeptide: AP-1 polypeptide complex. Thus,the present invention may be used to design drugs, including drugs witha capacity to inhibit binding of NF-AT_(c) to form NFAT.

Particularly preferred variants are structural mimetics of a dominantnegative NF-AT_(c) mutants, such as a polypeptide consisting essentiallyof amino acids 1-418 of FIG. 12 and substantially lacking amino acidscarboxy-terminal to residue 418. Such mimetics of dominant-negativemutant polypeptides can have substantial activity as antagonists orpartial agonists of NF-AT activation (and hence T cell activation).

8.6. Candidate Immunosuppressants

While any of the standard pharmaceutical sources of therapeuticcandidate agents may be used, a preferred class of agents suitable foruse in the screening assays of the present invention are macrolides,particularly those exhibiting a twisted amide peptidyl prolyl bond. See,Schreiber, Science, 251, 283-287 (1991). These agents are alsopreferably capable of binding to and blocking the cytosolic receptorsFKBP-12 and FKBP-13. See, Jin et al., Proc. Natl. Acad. Sci., U.S.A.,88, 6671-6681 (1991).

Agent screening using the methods of the present invention can befollowed by biological testing to determine if the agent has the desiredactivities in vitro and in vivo. The ultimate therapeutic agent may beadministered directly to the host to be treated or administered toexplanted cells which may then be returned to the host. Therapeuticformulations may be administered in any conventional dosage formulation.While for the active ingredient may be administered alone, preferably,it is presented as a pharmaceutical formulation. Formulations compriseat least one active ingredient as defined above together with one ormore pharmaceutically acceptable carriers thereof. Each carrier must beboth pharmaceutically and physiologically acceptable in the sense ofbeing compatible with the other ingredients and not injurious to thepatient. Formulations include those suitable for oral, rectal, nasal, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration of a therapeutically effective dose. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy.

9. Methods for Forensic Identification

The NF-AT_(c) polynucleotide sequences of the present invention can beused for forensic identification of individual humans, such as foridentification of decedents, determination of paternity, criminalidentification, and the like. For example but not limitation, a DNAsample can be obtained from a person or from a cellular sample (e.g.,crime scene evidence such as blood, saliva, semen, and the like) andsubjected to RFLP analysis, allele-specific PCR, or PCR cloning andsequencing of the amplification product to determine the structure ofthe NF-AT_(c) gene region. On the basis of the NF-AT_(c) gene structure,the individual from which the sample originated will be identified withrespect to his/her NF-AT_(c) genotype. The NF-AT_(c) genotype may beused alone or in conduction with other genetic markers to conclusivelyidentify an individual or to rule out the individual as a possibleperpetrator.

In one embodiment, human genomic DNA samples from a population ofindividuals (typically at least 50 persons from various racial origins)are individually aliquoted into reaction vessels (e.g., a well on amicrotitre plate). Each aliquot is digested (incubated) with one or morerestriction enzymes (e.g., EcoRI, HindIII, SmaI, BamHI, SalI, NotI,AccI, ApaI, BglII, XbaI, PstI) under suitable reaction conditions (e.g.,see New England Biolabs 1992 catalog). Corresponding digestion productsfrom each individual are loaded separately on an electrophoretic gel(typically agarose), electrophoresed, blotted to a membrane by Southernblotting, and hybridized with a labeled NF-AT_(c) probe (e.g., afull-length human NF-AT cDNA sequence of FIG. 12). Restriction fragments(bands) which are polymorphic among members of the population are usedas a basis to discriminate NF-AT_(c) genotypes and thereby classifyindividuals on the basis of their NF-AT_(c) genotype.

Similar categorization of NF-AT_(c) genotypes may be performed bysequencing PCR amplification products from a population of individualsand using sequence polymorphisms to identify alleles (genotypes), andthereby identify or classify individuals.

The following examples are offered by way of example and not by way oflimitation. Variations and alternate embodiments will be apparent tothose of skill in the art.

EXPERIMENTAL EXAMPLES Example 1 NF-AT is Enriched in Activated T Cells

A DNA binding assay was used to determine the amount of NF-AT present innuclear extracts from several stimulated and unstimulated cell lines. Aradiolabelled oligonucleotide probe corresponding to the NF-AT bindingsite was hybridized to concentrated nuclear extracts to determine theamount of NF-AT DNA binding activity present.

Procedure

Nuclear extracts were made according to the procedures of Ohlsson andEdlund (Cell 45:35 (1986)). Briefly, nuclei were extracted with 0.3 M(NH₄)₂SO₄ and the fraction that contained the nuclear proteins wasprecipitated with 0.2 g/ml (NH₄)₂SO₄ and dialyzed for 4 h. at 4° C. TheNF-AT binding site of the IL-2 enhancer (−290 and −263) was used as aprobe for binding activity. The binding experiment was carried outessentially as described in Shaw, J. P. Science 241:202-205 (1988)).

Results

As shown in FIG. 4, using a simple gel mobility shift assay, a complexforms with the NF-AT DNA-binding sequence and proteins present innuclear extracts of activated T cells but not with extracts ofnon-activated T cells or other types of cells. Only the nuclear extractfrom the Jurkat T cell line that had been stimulated with the T.cellactivating agents PMA and PHA contained detectable amounts ofNF-AT-specific DNA binding activity.

Example 2 Protein Synthesis is Required for Production of the NuclearComponent of the NF-AT Complex, While the Cytoplasmic Component isPreexisting

To determine whether protein synthesis is required for formation of thenuclear and cytoplasmic components of the NF-AT complex, or whether theproteins are constitutively present in the cells, an NF-AT-specific DNAbinding assay was done using NF-AT complex that had been reconstitutedfrom nuclear and cytoplasmic extracts from cells that had been activatedin the presence or absence of a protein synthesis inhibitor.

Procedure

NFATZ Jurkat cells were pretreated with 100 μM anisoymycin (Sigma), aprotein synthesis inhibitor, for 30 minutes before stimulating thecells. Conditions for activating the cells and preparing nuclear andcytoplasmic extracts are described in Example 3.

Results

As shown in FIG. 5, the protein synthesis inhibitor completely blockedthe appearance of the NF-AT complex in activated T cells (Lanes 2,4,6).This demonstrates that the nuclear component of the NF-AT complex issynthesized de novo upon activation of the T cells. In contrast,cytoplasmic extracts prepared from cells grown in the presence orabsence of a protein synthesis inhibitor were able to reconstitute theNF-AT complex (Lanes 3-6). Thus, the cytoplasmic component of the NF-ATcomplex preexists in the cells prior to stimulation, and additional denovo protein synthesis of NF-AT_(c) is not required.

Since the activation of the interleukin-2 gene as well as most early Tcell activation genes also requires protein synthesis, theseobservations are consistent with a prominent role for NF-AT in earlygene activation.

Example 3 NF-AT Can be Reconstituted from Cytosolic and Nuclear Subunits

A possible interpretation of the data presented in FIG. 5 is that NF-ATis synthesized but sequestered or compartmentalized within the cell andupon breakage of the cells some transcriptionally active NF-AT isformed. To test this hypothesis, the DNA binding ability of NF-ATcomplexes reconstituted from cytosolic and nuclear extracts fromstimulated and non-stimulated T cells, as well as from cells that hadbeen treated with FK506 just prior to stimulation was tested.

Procedure

NFATZ Jurkat cells and JK12/90.1 cells (a gift from N. Shastri) werestimulated for 2 hours with 20 ng/ml PMA and 2 μM ionomycin. Toquantitatively block NF-AT formation, FK506 100 ng/ml or CsA (Sandoz)500 ng/ml were used five minutes prior to the addition of PMA andionomycin, without any toxic effects to the cells. Nuclear extracts wereprepared as described previously with modifications. Cytoplasmicextracts were made from the same cells as the nuclear extracts.Following lysis of the cells with buffer A [10 mM Hepes (pH 7.8), 15 mMKcl, 2 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF] plus 0.05% NP-40,and pelleting of the nuclei, the cytoplasmic fraction was removed andstabilized with 10% (vol/vol) glycerol and 1/10 volume of buffer B [0.3M Hepes (pH 7.8), 1.4 M Kcl, and 30 mM MgCl₂]. The cytoplasmic extractwas centrifuged at 200,000 g for 15 minutes. An equal volume of 3 M(NH₄)₂SO₄ (pH 7.9) was added to the supernatant, and precipitatedproteins were pelleted at 100,000 g for 10 minutes. The pelletedcytoplasmic proteins were resuspended in buffer C [50 mM Hepes (pH 7.8),50 mM KCl, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF, 10% (vol/vol) glycerol]and desalted by passage over a P6DG column (BioRad). Proteinconcentrations were determined using a BioRad protein assay kit. Toassess the completeness of the nuclear and cytoplasmic fractionation, weassayed for Oct-1 (a constitutive nuclear located DNA binding protein)binding activity and β-galactosidase (cytoplasmic localized) enzymeactivity. We found no Oct-1 binding activity in the cytoplasmic fractionand found that β-galactosidase activity is present in the cytosol at3.7-fold higher concentration than in the nuclear fraction (data notshown). Electrophoretic mobility shift assays were done essentially asdescribed. Fried, M. and D. M. Crothers NAR 9:6505-6526 (1981). Bindingreactions were carried out as previously described. Fiering, S. et al.Genes Dev. 4 1823-1834 (1981). Total amount of protein used in eachbinding reaction was 10 μg. The end-labelled binding site for NF-AT wasderived from the human IL-2 enhancer (−285 to −255 bp). Theoligonucleotide sequence is 5′-gatcGGAGGAAAAACTGTTCATACAGAAGGGGT-3′ (SEQID NO: 61). The mutant NF-AT probe essentially differs from the NF-AToligonucleotide at four contact guanosine residues. The sequence is5′-gatcAAGAAAGGAGtAAAAAaTtTTTaATACAGAA-3′(SEQ ID NO: 62). Lower caseletters indicate mutated residues. Competition with 10 ng of unlabeledoligonucleotide represents a 100- to 200-fold molar excess over labeledprobe.

Results

In the nuclear extracts prepared from stimulated/FK506-treated cells,NF-AT binding activity is reduced substantially and is not observed inthe cytoplasmic fractions (FIG. 6A, lanes 3 and 6). Remarkably, bindingactivity was completely reconstituted by mixing nuclear extracts fromstimulated/FK506-treated cells together with cytoplasmic fractions fromnonstimulated, or stimulated/FK506-treated cells, neither of which haveNF-AT binding activity (FIG. 6A, lanes 7 and 9). Although the mobilityof the reconstituted DNA-protein complex is slightly faster than thecharacteristic mobility of the NF-AT complex, DNA binding specificity isidentical. (FIG. 6B, lanes 4-9). Nuclear extracts from nonstimulatedcells are not complemented by any of the cytoplasmic extracts (FIG. 6Blanes 1-3) suggesting that stimulation of the cells is essential forsynthesis of the nuclear component of NF-AT.

While cytoplasmic extracts from nonstimulated andstimulated/FK506-treated cells can reconstitute the NF-AT complex,cytoplasmic extracts from stimulated cells show only partialreconstitution of NF-AT binding activity (FIG. 6A, lane 8) implying thatthe cytoplasmic component of NF-AT preexists in nonstimulatedcytoplasmic extracts and is translocated to the nucleus followingstimulation in the absence of FK506.

We used rapamycin as a control for non-specific effects of FK506.Rapamycin is a structural analog of FK506, and like FK506, contains astructural mimic of a twisted leucyl-prolyl amide bond, binds FK-BP, andinhibits its isomerase activity (Bierer et al. (1990) Science 250:556;Bierer et al. (1990) PNAS(USA) 87:9231; and Rosen et al. (1990) Science248:863). Despite the fact that rapamycin inhibits isomerase activity,it antagonizes the actions of FK506 on NF-AT-directed transcription,IL-2 gene activation, T cell activation, and programmed cell death(Bierer et al. (1990) PNAS 87:9231; Dumont et al. (1985) J. Immunol.134:1599; and Dumont et al. (1990) J. Immunol. 144:1418). Rapamycin didnot block translocation of the cytoplasmic component of NF-AT to thenucleus following activation (FIG. 6A, lane 10). This is consistent withits failure to block NF-AT directed transcription (Mattila et al.,supra).

To determine if impaired nuclear import is also a property of theimmunosuppressive prolyl isomerase inhibitor CsA, we teeted the effectsof CsA. The biological effects of FK506 and CsA on the immune responseare essentially identical (Karrtunen et al. (1991) PNAS 88:3872). CsAcompletely blocks NF-AT directed transcription in T cells and extractsof cells stimulated in the presence of CsA contain less NF-AT bindingactivity than stimulated controls (Emmel et al. (1989) Science246:1617). Accordingly, mixing nuclear extracts from stimulatedCsA-treated cells with cytoplasmic extracts from the sane cells ornonstimulated cells reconstitutes NF-AT binding activity (FIG. 6C, lanes4-6). Again, nonstimulated nuclear extracts are not able to becomplemented by any cytoplasmic extract (FIG. 6C, lanes 1-3). Thus,these results suggest that CsA and FK506 both block the translocation ofa pre-existing cytoplasmic component which constitutes part of the NF-ATDNA binding complex.

Example 4 The Cytosolic Form of NF-AT (NF-AT_(c)) is SelectivelyExpressed in T Cells

Despite the fact that the actions of CsA and FK506 are tissue specific,their binding proteins are ubiquitous (Koletsky et al. (1986) J.Immunol. 137: 1056; Kincaid et al. (1987) Nature 330:176; Sieklerka etal. (1989) Nature 341:755; and Harding et al. (1989) Nature 341:758).This apparent quandary could be rationalized if the drug-isomerasecomplex acted on a T cell specific molecule. To determine whether thecomponents of the NF-AT complex are found in cell types other than Tcells, we tested whether nuclear or cytoplasmic extracts of HeLa cellscan be used to reconstitute NF-AT complex alone or in conjunction withextracts from Jurkat cells.

Procedure

HeLa S3 cells were grown in spinner flasks at 37° C. in S-MEM(Gibco-BRL) supplemented with 5% fetal calf serum, penicillin (100U/ml), and 100 μg/ml of streptomycin. HeLa S3 were stimulated, nuclearand cytoplasmic extracts were prepared, and gel mobility shift assayswere carried out under conditions identical to those described in FIG.6.

Results

HeLa cytoplasmic extracts do not contain NF-AT and homologous mixing ofnuclear and cytoplasmic extracts do not contain NF-AT and homologousmixing of nuclear and cytoplasmic extracts from HeLa cells failed toreconstitute NF-AT binding activity (FIG. 7A, lanes 1-9). In contrast,heterologous mixing of Jurkat cytoplasmic extracts with nuclear extractsfrom HeLa cells reconstituted NF-AT binding activity (FIG. 7B, lanes1-3). Furthermore, the reconstituted NF-AT binding activity is specificas demonstrated by oligonucleotide competition (FIG. 7B, lanes 4-9).These results suggest that the oligonucleotide competition (FIG. 7B,lanes 4-9). These results suggest that the nuclear component of NF-AT ispresent in HeLa cells. In contrast, HeLa cell cytoplasmic extractscannot reconstitute NF-AT binding activity when mixed with nuclearextracts from stimulated/FK506-treated Jurkat cells (FIG. 7C, lanes 1-3)implying that the cytoplasmic component is T cell specific while thenuclear component of NF-AT is not.

Example 5 Nuclear Import of the Cytosolic Component can be Induced withIonomycin While Synthesis of the Nuclear Component Requires Only PMA

A unifying feature of the actions of FK506 and CsA is that they inhibitprocesses which require Ca²⁺ mobilization (Mattila et al. (1990) EMBO J.9:4425; Kay et al. (1989) Cell. Immun. 124:175; Cirillo et al. (1990) J.Immunol. 144:3891; Gunter et al. (1989) J. Immunol. 142:3286). Inductionof NF-AT binding and transcriptional activity requires physiologicstimuli that are believed to be mimicked by agents that increaseintracellular Ca²⁺ and activate protein kinase C (PKC) (Shaw et al.(1988) Science 241:202).

To examine the requirements for induction of the nuclear and cytoplasmicsubunits of NF-AT, extracts were prepared from cells stimulated witheither PMA alone or ionomycin alone and tested for their ability toreconstitute DNA-binding activity.

Procedure

Gel mobility shifts and preparation of nuclear and cytoplasmic extractswere carried out as described in Example 3.

Results

Cytosolic extracts from ionomycin-treated cells show less ability toreconstitute DNA binding when added to nuclear extracts of stimulatedFK506-treated cells than either cytosolic extracts from non-stimulatedcells, cytosolic extracts from PMA-stimulated cells or cytosolicextracts from cells stimulated with both PMA and ionomycin (FIG. 8).FK506 treatment did not inhibit PMA/ionomycin-stimulated cells fromsynthesizing the nuclear component of NF-AT (FIG. 8, Lanes 9-12).Furthermore, mixing cytoplasmic extracts from PMA-stimulated orionomycin-stimulated cells with nuclear extracts fromstimulated/FK506-treated cells fail to reconstitute NF-AT DNA-bindingactivity (FIG. 8, Lanes 4 and 7), suggesting that the preexistingcytoplasmic subunit translocated to the nucleus. Thus, CsA and FK506appear to inhibit the Ca²⁺-dependent translocation of the cytoplasmiccomponent of NF-AT.

Example 6 FK506 does not Inhibit NF-AT-Dependent Transcription In Vitro

The effect of FK506 on the ability of NF-AT to direct transcription wastested by preparing nuclear extracts from stimulated orstimulated/FK506-treated cells and testing their ability to transcribe aG-less cassette in which transcription was dependent upon three NF-ATsites located within a synthetic promoter.

Procedure

Promoter constructs, nuclear extracts and transcription reactions wereprepared as described. NFATZ Jurkat cells, derived from a human T cellleukemia, were stimulated with 20 μg/ml PMA (Sigma), 2 μM ionomycin(Calbiochem) for 2 hours. FK506 was used at 10 ng/ml and added fiveminutes prior to the addition of PMA and ionomycin. Ribonucleaseprotection assay of the NF-AT/lacZ mRNA was carried out as previouslydescribed. Transcription was quantitated using a radioanalytic imagingsystem (AMBIS).

Using the human Jurkat T cell line (Wiskocii et al. (1985) J. Immunol.1599), we developed an activation-dependent, T cell specific in vitrotranscription system which faithfully reflects the complex requirementsfor IL-2 transcription and more generally T cell activation.

In Vitro Transcription Protocol:

Procedure

(i) Cell Culture and Stimulation Conditions

Jurkat cells were grown in RPMI 1640 without L-glutamine, 8% fetal calfserum (FCS) (Irvine Scientific), with penicillin (100 units/ml) andstreptomycin sulfate (100 μg/ml) at 5% CO₂ concentrations. Cells weresplit 1:3 thirty-six hours before stimulation. The morning of thestimulation, the Jurkat cells (1×10⁶ cells/ml) were centrifuged at 3500rpm (2000×g), in a GS-3 rotor for 10 minutes and then resuspended infresh media to a concentration of 2×10⁶ cells/ml. In general, 2 μMionomycin (calbiochem) and 20 ng/ml PMA (Sigma) were used to stimulatethe cells. During the 2 hour stimulation, the cells were constantlyshaking to prevent the layering of cells on the bottom of the flask.

Hela S3 cells were grown in S-MEM (Gibco) with 8% GCS, with penicillin(100 units/ml) and streptomycin sulfate (100 μg/ml) and 2 mML-glutamine. Hela S3 were stimulated with 20 ng/ml PMA and 2 μMionomycin.

(ii) Plasmid Construction

The IL-2 G-less plasmid was constructed by fusing the IL-2 enhancer(−326 to +24) to a 377 base pair (bp) G-less cassette generouslyprovided by R. Roeder (Sawadogo and Roeder, 1985) using polymerase chainreaction overlap extension techniques (Horton, R. M. et al. Gene77:61-68 1989; Ho, S. N., et al. Gene 77:51-59 1989). The IL-2 enhancerG-less cassette contained on a Xho I-Bam HI fragment was inserted into apUC derivative containing an Xho I site in the polylinker. To avoid PCRartifacts the entire IL-2 enhancer G-less cassette was sequenced. Thetotal size of the IL-2 enhancer G-less transcript is 401 nucleotides(nt). The NF-AT multimer which contains 3 NFAT binding sites (−286 to−257) and NF-IL-2A multimer which contains 4 NF-IL-2A binding sites (−94to −65) G-less constructs were made by digesting pE3.1 and pA4.1 (Durandet al. 1988) with Asp 718 and Bam HI, respectively, and ligating thefragments into an Asp 718-Bam El digested τ-fibrinogen G-less cassetteconstruct. τ-fibrinogen G-less was constructed by fusing −54 to +1 ofthe τ-fibrinogen promoter (Crabtree, G. R. and Kant, J. A. Cell31:159-166 1982; Durand et al. 1987) to the 377 bp G-less cassette usingPCR overlap extension techniques. All regions of the construct madeusing PCR technology were sequenced to avoid any point mutations usingSequenase DNA sequencing kit (U.S. Biochemical). The τ-fibrinogenpromoter is a minimal promoter containing only a Spl binding and TATAbox. Between +1 of the τ-fibrinogen promoter and the G-less cassette aSsp I restriction enzyme site was inserted. Both the ARRE-2 and ARRE-1G-less constructs generate 383 nt transcripts.

The HNF-1 (hepatocyte nuclear factor 1) G-less plasmid was constructedby inserting tandemly linked NF-I binding sites from Rat β-fibrinogenpromoter (−77 to −65) (Courtois et al. 1987) into Xho-Sal polylinkersites in τ-fibrinogen G-less construct. The adenovirus major latepromoter (AdMLP) G-less construct was a generous gift of Drs. M.Sawadoga and R. Roeder. Total size of the AdMLP G-less transcript is 280nt.

(iii) Preparation of Nuclear Extracts

Jurkat and liver in vitro transcription nuclear extracts wereessentially made as described by Gorski et al. (Gorski et al. 1986;Maire et al. 1989) with some exceptions. First, the cells were broken in1.5 M sucrose-glycerol solution to reduce the amount of frictional heatgenerated during cell lysis. Second 0.5% (vol/vol) nonfat dry milk wasadded to the homogenization buffer as had been previously described(Maire et al. 1989). Third, the Jurkat nuclei were fractionated on onlyone 2.0 M sucrose pad preceding salt extraction. Briefly, allmanipulations were performed in the cold, and all solutions, tubes, andcentrifuges were chilled to 4° C. Protease inhibitors, antipain (1μg/ml), leupeptin (1 μg/ml), 0.1 mM phenylmethylsulfonyl fluoride (PMSF)and 0.1 mM benzamidine, were added to all buffers except the dialysisbuffer. One mM dithiothreitol was added to all buffers. Followingstimulation in the case for Jurkats, the cells (10⁹) were centrifuged ina GS-3 rotor, 3500 rpm (2000×g), for 10 minutes. The media was pouredoff and the cells were rinsed with 40 mls of phosphate buffered saline.Resuspended pellets were then centrifuged 1000 rpm (200×g), 10 minutesin a \Beckman GPR tabletop centrifuge. The cell pellet was resuspendedin 10 ml of homogenization buffer (10 mM Hepes [pH 7.6] 25 mM KCl, 0.15mM spermine, 0.5 mM spermidine, 1 mM EDTA, 1.25 M sucrose, 10% glycerol(vol/vol), 0.5% nonfat dry milk (vol/vol). An aqueous 0.1 g/ml nonfatdry milk solution was centrifuged for 10 minutes in a SS-34 rotor at1000 rpm (11950×g) to remove undissolved milk solids before adding toany solution.

The cells were dounced (Teflon-glass homogenizer) until broken using a ½hp drill press (Jet Tools Inc) at high speed. Cells were checked forlysis. Generally, greater than 80% of the cells were lysed. Followinglysis, 46 mls of 2M sucrose homogenization buffer (10 mM Hepes (pH 7.6],25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2M sucrose,10% glycerol (vol/vol), 0.5% nonfat dry milk (vol/vol) were added to thedounced cells. The homogenized cells (28 mls) were layered on to 10 mlsucrose pads (10 mM Hepes (pH 7.6), 25 mM KCl, 0.15 mM spermine, 0.5 mMspermidine, 1 mM EDTA, 2M sucrose, 10% glycerol (vol/vol) andcentrifuged at 24,000 rpm for 60 minutes in a SW 28 rotor (103,000×g).

The pelleted nuclei were resuspended in a total of 6 ml of nuclear lysisbuffer (10 mM Hepes [pH 7.6], 100 mM KCI, 3 mM MgCl₂, 0.1 mM EDTA, 10%glycerol(vol/vol).) One ninth volume of 3M (NH₄)₂SO₄ pH 7.9 was addedand mixed constantly for 30 minutes. The viscous lysate was centrifuge40,000 rpm, 60 minutes, in a Ti 50 rotor (150,000×g) to pellet thechromatin.

Following centrifugation, the tubes were quickly removed and thesupernatant transferred to another tube before the pelleted chromatinbegan to reswell. To the supernatant, 0.3 grams of solid (NH₄)₂SO₄ perml were added. The tube was gently mixed for 10 minutes or until all the(NH₄)₂SO₄ had gone into solution. The tubes were placed on ice for 40minutes and gently mixed every 10 minutes. The precipitated proteinswere then centrifuged for 15-20 minutes, 40,000 rpm, in a Ti 50 rotor(150,000×g). At this point, the pellet was immediately resuspended indialysis buffer (25 mM Hepes [pH 7.6], 40 mM KCl, 0.1 mM EDTA, 10%glycerol (vol/vol).) Protein extracts from 1×10⁹ Jurkat cells wereresuspended in 200-300 μl of dialysis buffer resulting in a finalprotein concentration of 10 mg/ml. Extracts were dialyzed twice for 2hours in the cold against 100 volumes of dialysis buffer. Duringdialysis a precipitate forms that at the end of dialysis was removed bycentrifugation in a microfuge (Brinkman Instruments) at a setting of 14for 5 minutes. Protein concentrations were determined with a Bio-Radprotein assay kit using BSA as a standard. Protein extracts were frozenin small aliquots on dry ice and immediately stored in liquid nitrogen.

HeLa S3 nuclear extracts were made as previously described (Shapiro, D.J. et al. DNA 7:44-45 1988).

(iv) Transcription Reactions

In general, transcription reactions (20 μl) contained 40 μg/ml ofcircular DNA template [400 ng of the test construct, 40 ng of the AdMLPG-less construct, and 360 ng of poly dI-dC (Pharmacia)] and between 3-5mg/ml nuclear protein extract in a buffer containing 25 mM Hepes (pH7.6), 50 mM KCl, 6 mM MgCl2, 0.6 mM each of ATP and CTP, 7 μM UTP, 7 μCi[a-³²P] UTP (Amersham, 400 Ci/mmole), 0.5 mM 3′-O-methyl GTP(Pharmacia), 150 units of RNase T1 (BRL), 12 unites of RNase inhibitor(Amersham) and 12% glycerol (vol/vol). EDTA and DTT were contributed bythe extract. Transcription reactions using liver or HeLa nuclearextracts contained 40 μg/mil of circular DNA templates (400 ng of thetest construct and 400 ng of the AdMLP). All other reaction conditionswere kept constant. The reactions were incubated for 45 minutes at 30°C. The transcription reactions were terminated by adding 280 μl of stopbuffer (50 mM Tris-HCl [pH 7.6], 1% SDS, 5 mM EDTA) and were extractedtwo times with phenol and one time with chloroform. The RNA wasprecipitated with 15 μg of glycogen, 0.3M sodium acetate (pH 5.2) and2.5 volumes of ETOH. The pellets were rinsed with 70% ethanol, airdried, and resuspended in 10 μl of loading dye (90% formamide, 0.01%xylene cyanol, and 0.01% bromophenol blue in 1×TBE.) The transcriptswere analyzed on 6% denaturing polyacrylamide gels. In general, the gelswere exposed overnight at room temperature using XAR-5 (Kodak) x-rayfilm. Normalized fold induction is calculated by normalizing the amountof transcription from the test G-less construct to that observed fromthe AdMLP G-less construct and then dividing the amount of test G-lesstranscription from stimulated nuclear extracts by the amount of testG-less transcription from nonstimulated nuclear extracts. Autoradiogramswere quantitated using an Ambis radioanalytic imaging system (AmbisSystems, San Diego, Calif.).

Results

Surprisingly, nuclear extracts from Jurkat cells that had beenstimulated for 2 hours with PMA and ionomycin in the presence of FK506(10 ng/ml) transcribe the IL-2 G-less template at levels nearlyequivalent to extracts from fully stimulated Jurkat cells (FIG. 9A,compare lanes 2 and 3) even though transcription of the endogenous IL-2gene in these cells is fully inhibited (data not shown). Since most ofthe inhibitory effects of CsA and FK056 on IL-2 gene activation havebeen shown to be due to the inhibition of NF-AT function (Emmel supra;Mattila et al., supra), we also examined transcription directed by thisprotein. In vitro transcription directed-by multimerized binding sitesfor the NF-AT protein was reduced 2.5-fold in nuclear extracts ofstimulated/FK506-treated cells (FIG. 9A, compare lanes 5 and 6) despitethe fact that NF-AT dependent transcription was totally blocked in thecells used to prepare the extracts (FIG. 9B). In these extracts, NF-ATDNA-binding activity is reduced about 50 to 80% far less than theinhibitory effects on in vivo IL-2 gene expression that are generally inexcess of 99% (Mattila et al., supra), but commensurate with the effectson NF-AT dependent in vitro transcription. Thus, it appears thatstimulated/FK506-treated cells contain a reduced amount of NF-AT thatfunctions in vitro but not in vivo.

Example 7 Tandem NF-AT Binding Sites Direct Expression of T Antigen toActivated Lymphocytes in Transgenic Mice

To determine whether a transcriptional promoter under the control ofNF-AT regulatory sites will specifically direct expression of a linkedgene to activated lymphocytes, we utilized a cell line that contains aconstruct in which tandem NF-AT binding sites are linked upstream of agene encoding T antigen (Verweij et al., J. Biol. Chem. 265:15788-15795).

Procedure

Total RNA was isolated from various tissues and cells using guanidiumthiocyanate and hot phenol extraction. Equal amounts (10 μg)of RNA wereused. RNA mapping experiments with the Sp6/T7 RNA polymerase system weredone according to (Melton, D. A. NAR 12:7035-7056 (1984)). For mappingcorrectly initiated NF-AT-Tag mRNA, a SP6 RNA probe was transcribed fromEco RI digested pSP6IL-2 vector containing a 117 bp Xho I-Hind IIIfragment (−70 to +47 of NFAT-Tag). Hybridization was allowed to proceedat 42° C. for 16 h and samples were digested with 4 ug/ml RNase A and160 unit s/ml RNase T1 at 30° C. for 1 h. Protected fragments were runon a 5% denaturing polyacrylamide gel and exposed to XAR-5 film.

Results

As shown in FIG. 10, only lymphoid cells transcribed the T antigen genewhich was under the control of the tandem NF-AT binding sites. Thus, anarray of NF-AT binding sites is useful for directing expression of alinked gene specifically to activated lymphoid cells.

Example 8 Activation of NF-AT Probably Requires Phosphorylation

Evidence for phosphorylation of NF-AT was obtained by treating nuclearextracts with calf alkaline phosphatase and examining the mobility ofthe NF-AT DNA-binding complex on nondenaturing gels. As shown in FIG.11, dephosphorylation of NF-AT with calf intestinal phosphatase reducesits ability to associate with its DNA binding site (FIG. 11, lane 4).

Conclusion: Model for the Actions of FK506 and Cyclosporin A: Their Rolein Preventing Nuclear Import of NF-AT:

A calcium stimulus induced by the antigen leads to the nuclear import ofa subunit of NF-AT. Once in the nucleus, the cytosolic subunit combineswith a newly induced nuclear subunit to produce a complex having bothDNA-binding activity and transcriptional activity. Neither subunit alonehas DNA binding activity and neither subunit alone has transcriptionalactivity. Cyclosporin A and FK506 prevent the import of the cytosoliccomponent of NF-AT by either preventing the development of competencefor nuclear transfer of the cytosolic component of NF-AT or by blockingthe appearance of nuclear import signals for NF-AT.

Example 9 Determination of the Nucleotide and Amino Acid Sequence ofHuman NF-AT_(c) cDNA

This example represents the isolation and purification of this novelhuman NF-AT protein, NF-AT_(c), the determination of the amino acidsequence of its fragments and the isolation and sequencing of the cDNAclone encoding this protein.

Since our previous work indicated that the cytosolic component of NF-ATwas present at relatively low concentrations in human lymphoid celllines (Northrop et al. (1993) J. Biol. Chem. 268: 2917-2923), we choseto purify NF-AT_(c) from bovine thymus. Accordingly, the protein waspurified from bovine thymus glands obtained from newborn calves.Approximately 20 bovine thymuses were homogenized to make a cytosolicextract which was then subjected sequentially to 1) ammonium sulfateprecipitation, 2) sulphopropyl Sepharose chromatography, 3) heparinagarose chromatography, 4) affinity chromatography using a multimerizedbinding site for NF-AT, with the sequence5′-ACGCCCAAAGAGGAAAATTTGTTTCATACA-3′(SEQ ID NO: 39) coupled to sepharoseCL4B, and 5) HPLC on a reverse phase C4 column. The resulting purifiedprotein was subjected to cleavage with LysC/ArgC and fragments isolatedby HPLC. The sequences of these individual fragments were thendetermined by automated Edman degradation. Sequences obtained included:LRNSDIELRKGETDIGR (SEQ ID NO: 35) and LRNADIELR (SEQ ID NO: 40).Degenerate oligos corresponding to GETDIG (SEQ ID NO: 41) (reverseprimer) and RNADIE (SEQ ID NO: 42) (forward primer) were made. Thedegenerate oligo PCR primers had the following sequences:

(SEQ ID NO: 43) A forward: (A/C)GIAA(C/T)GCIGA(C/T)AT(A/C/T)GA(A/G) (SEQID NO: 44) A reverse: ICC(A/G/T)AT(A/G)TCIGT(C/T)TCICC

To isolate the cDNA, oligonucleotide probes were made corresponding tothe determined amino acid sequence and used as PCR primers to isolate a45 base fragment from bovine cDNA prepared from the bovine thymus. Thebovine PCR product comprised the nucleotide sequence CTG CGG AAA whichencodes -L-R-K-. The same 45 bp fragment can be amplified from human andmouse sources.

This bovine PCR product was then used to screen a cDNA library of thehuman Jurkat T cell line. Clones were isolated at frequencies of about 1in 100,000 to 1 in 200,000. A total of five human cDNA clones of variouslengths were isolated. Two overlapping clones, one containing the 5′ endand one containing the 3′ end were ligated together using a unique EcoRIrestriction site present in each clone, to produce a full-length cDNAwhich corresponded in length to the messenger RNA determined by Northernblotting.

The sequence of the NF-AT_(c) cDNA was determined by the Sanger methodand the complete nucleotide and predicted amino acid sequence is shownin FIG. 12. The nucleotide sequence contained 2742 nucleotides and isshown in FIG. 12 (SEQ ID NO: 45). The cDNA encodes a protein of 716amino acids having the amino acid sequence shown in FIG. 12 (SEQ ID NO:38) with a predicted molecular weight of 77,870. An in-frame stop codonupstream from the initiator methionine indicates that the entireNF-AT_(c) protein is encoded by this cDNA. The initiator methionineindicated in FIG. 12 was determined by fusing this reading frame to aglutathione transferase gene and transfecting the resultant clone intobacteria. The resultant clone produced a fusion protein of the propermolecular weight, indicating that the reading frame designated with theinitiator methionine is indeed the correct reading frame. The positionof the stop codon was determined by a similar procedure. In addition,the stop codon corresponds to the reading frame for nine of thedetermined amino acid sequences. A unique repeated sequence of 13residues was also identified.

The total NF-AT_(c) protein structure was aligned against individual Relproteins using a MacIntosh shareware program called DOTALIGN utilizingthe alignment parameters of the FASTA programs. Significant homology wasobserved that corresponded to the Rel domains of these proteins.Enhanced amino acid residue alignment was done using ALIGN from the samesuite of programs. Alignment of the Rel similarity regions of NF-AT_(c)and NF-AT_(p) was done by hand with no insertions necessary, The Miyataalphabet (Miyata et al. (1979) J. Mol. Evol. 12: 214-236) was used todetermine similar residues. FIG. 15 shows results of such sequencealignments.

The carboxy-terminal half of NF-AT_(c) shows limited similarity to theDNA binding and dimerization regions of the Dorsal/Rel family oftranscription factors (FIG. 4, for review, Nolan and Baltimore (1992)Current Biology, Ltd. 2: 211-220) however, NF-AT_(c) appears to be themost distantly related member of the group. There are a significantnumber of amino acid changes resulting in charge reversals between theRel family members and NF-AT_(c), suggesting that charge might beconserved at these positions to maintain salt bridges. Six additionalpeptides obtained from the purified bovine protein are derived from thebovine homolog of NF-AT_(p), a cDNA fragment of which was reported byMcCaffrey et al. (1993) Science 262: 750-754). Comparison of NF-AT_(c)and NF-AT_(p) reveals that they are products of distinct genes with 73%amino acid identity in the Rel similarity region (FIG. 15), however,there is very little similarity outside this region. A murine cDNA forNF-AT_(c) was isolated and the predicted protein was found to be 87%identical to human NF-AT_(c), and distinctly different from murineNF-AT_(p).

Example 10 Expression of NF-AT_(c) in T and non-T cells

The cDNA shown in FIG. 12 was fused to the Hemophilus influenzahemaglutinin (HA) 12 amino acid epitope tag in the determined readingframe and operably linked to the SRα promoter in the vector pBJ5 (Lin etal, 1990, Science 249:677-679). The resultant construct was transientlytransfected by electroporation into Jurkat human T lymphocytes, and intoCos fibroblast cells. Expression of the epitope-tagged NF-AT_(c) proteinwas determined by Western blotting of whole cell extracts prepared fromthe transfected cells, using an antibody (12CA5, Berkeley Antibody Co.,CA) that detects the HA epitope.

FIG. 13 shows that NF-AT_(c) cDNA construct is able to express a proteinof appoximately 120 kDA corresponding precisely in size to that of thepurified protein, in both Jurkat T cells and Cos cells (see lanes 3 and6 labeled NF-AT*. Lane 2 shows as control, NF-AT without the epitope tagwhich cannot be detected in the Western blot).

Example 11 Transfection of NF-AT_(c) Activates Transcription in Both Cosand Jurkat Cells

The NF-ATc cDNA was operably linked to a portion of the SV40 early genepromoter and the mV transcription regulatory regions in the pBJ vector.This expression vector was co-tranfected into Jurkat and Cos cells witheither a) three copies of NF-AT binding site linked to and directingtranscription of luciferase (results shown in FIGS. 14A and 14B) theentire IL-2 enhancer/promoter directing transcription of luciferase(results shown in FIG. 14B). Cytosolic extracts were prepared andluciferase assays carried out by standard procedures (de Wet et al,1987, Mol. Cell. Biol. 7:724-837).

The results demonstrate that in both Cos cells and Jurkat cells,overexpression of the NF-AT_(c) protein dramatically enhancesNF-AT-dependent transcription by 50-1000 fold (see FIG. 14A). Inaddition, overexpression of the NF-ATc protein in Cos cells activatesthe IL-2 promoter, which in the absence of NF-AT_(c) cannot otherwise beactivated (see FIG. 14B).

These results indicate that the cDNA clone encodes a functionalNF-AT_(c) protein and that NF-AT_(c) is the protein which restrictsexpression of interleukin-2 to T cells.

Example 12 NF-AT_(c) mRNA and Protein Expression

NF-AT_(c) mRNA is absent in Hela cells (FIG. 16, panel A, lane 7), acell line incapable of IL-2 or NF-AT-dependent transcription, but isinducible in Jurkat cells (FIG. 16, panel A). This induction issensitive to cyclosporin A, (CsA), indicating that NF-AT_(c) mayparticipate in an auto-stimulatory loop as CsA has been shown to blockits nuclear association (Flanagan et al. (1991) Nature 352: 803-807).Two B cell lines, muscle tissue, Hep G2 cells and myeloid leukemia cellsdo not express NF-AT_(c) mRNA (FIG. 16 panel B). These observations areconsistent with the observed T cell-restricted pattern of IL-2transcription and NF-AT activity. Previous studies (Verweij et al.(1990) J. Biol. Chem 265: 15788-15795) revealed NF-AT-dependenttranscription predominantly in spleen, thymus and skin of transgenicmice expressing an NF-AT-dependent reporter gene. Consistent with theseobservations, murine NF-AT_(c) mRNA shows the same pattern of expression(FIG. 16 panel C). Small amounts of NF-AT_(c) expression are seen inlung and heart, however, this may be due to contamination withcirculating T cells. Murine NF-AT_(p) mRNA, also assayed by quantitativeribonuclease protection, was found to be expressed at approximatelyequal levels in brain, heart, thymus and spleen (FIG. 16 panel C). Incontrast to NF-AT_(c), NF-AT_(p) was not inducible by PMA and ionomycin(FIG. 16 panel C).

Methods: Specific human or mouse NF-AT_(c) or mouse NF-AT_(p) cDNAfragments were used as templates for the synthesis of RNA transcripts.Ribonuclease protection was done according to Melton et al. (1984) Nucl.Acids. Res. 12: 7035-7056) using 10 μg of total RNA. Splenocytes andthymocytes were isolated and treated as described (Verweij et al. (1990)J. Biol. Chem 265: 15788-15795) before isolating RNA, otherwise wholetissue was used.

Example 13 Functional Expression of NF-AT_(c)

NF-AT luciferase and IL-2 luciferase have been described (Northrop etal. (1993) J. Biol. Chem. 268: 2917-2923). β28 luciferase wasconstructed by inserting a trimerized HNF-I recognition site (β28) inplace of the NF-AT recognition sites in NF-AT luciferase. The plasmidpSV2CAT (Gorman et al. (1982) Mol Cell. Biol. 2: 1044-1050) was used asan internal control for transfection efficiency. Cells were transfectedwith 1.5 μg of luciferase reporter and 3 μg of expression construct asdescribed. After 20 hours of growth, cells were stimulated for 8 hrs.with 20 ng/mil PMA plus or minus 2 μM ionomycin, and harvested forluciferase (de Wet et al. (1987) Mol. Cell. Biol. 7: 725-737) and CATassays (Gorman et al. (1982) Mol. Cell. Biol. 2: 1044-1050).

Cos cells were transfected with epitope tagged NF-AT_(c) as described.Cos cells, Jurkat cells, and murine thymocytes were stimulated for 3 hrwith PMA and ionomycin. Hela cells were stimulated for 3 hr with PMAalone and nuclear extracts prepared as described (Fiefing et al. (1990)Genes & Dev. 4: 1823-1834). Cytosols were prepared from non-stimulatedCos cells. Gel mobility shifts were performed as previously described(Flanagan et al. (1991) Nature 352: 803-807; Northrop et al. (1993) J.Biol. Chem. 268: 2917-2923). Antisera were raised in mice immunized withbacterially expressed glutathione S-transferase fusion proteins usingthe vector pGEX-3X (Pharmacia) and purified on glutathione agarose.Fusion proteins contained NF-AT_(c) residues 12 to 143 (immune-1) and 12to 699 (immune-2).

NF-AT_(c), expressed in non T cell lines specifically activatedtranscription from the NF-AT site and the IL-2 promoter, (FIG. 17 panelA (left), and FIG. 17 panel B). In transiently transfected Jurkat cells,overexpression of NF-AT_(c) activated an NF-AT-dependent promoter butnot an HNF-1 dependent promoter (FIG. 6 panel A (right)) or anAP-1-dependent promoter. Transfection of the NF-AT_(c) cDNA gives riseto DNA binding activity that is indistinguishable from endogenous NF-AT(FIG. 17 panel C, lanes 1-4). Antibody directed against the HA epitopeencoded by the transfected cDNA induces a supershift of the NF-ATcomplex indicating that NF-AT_(c) participates in this activity. Thenuclear NF-AT activity in transfected Cos cells comigrates with, and hasthe same binding specificity as, the native nuclear complex in T-cells(FIG. 17 panel C, lanes 4-11). Cytosolic extracts from NF-ATc,transfected Cos cells can reconstitute NF-AT DNA binding activity whenmixed with Hela nuclear extract (FIG. 17 panel C, lanes 12-16) as docytosolic extracts from T-cells (Flanagan et al. (1991) Nature 352:803-807; Northrop et al. (1993) J. Biol. Chem. 268: 2917-2923). Antiseraraised against bacterially expressed fragments of NF-AT_(c) that have nosimilarity to NF-AT_(p) are able to induce a supershift of theendogenous NF-AT complex, but not the AP-1 complex, from Jurkat cells orthymocytes (immune-1 and immune-2 respectively, FIG. 17 panel D).Immune-2 antisera reduced the DNA-protein complex produced using murinethymic nuclear extracts significantly, consistent with the relativelyequal representation of NF-AT_(c), and NF-AT_(p) peptides in thepurified protein from bovine thymus.

Example 14 NF-AT_(c) Dominant Negative Mutant Assayed in TransientTransfection Assays

A dominant negative NF-AT_(c), prepared after extensive deletionanalysis of the cDNA, indicated that the amino terminal domain wouldblock NF-AT-dependent function without affecting binding. This region ofthe cDNA is not found in NF-AT_(p) and hence can be used to assess thecontribution of NF-AT_(c) to the activation of the IL-2 gene. Thedominant negative NF-AT_(c) used consists of a carboxy terminaltruncation of the epitope tagged NF-AT_(c) expression plasmid (supra)extending to the PvuII site at amino acid 463. Transfection of thisdominant negative resulted in more than 90% inhibition of IL-2 promoterfunction as well as transcription directed by the NF-AT site (FIG. 18).This effect was highly specific since transcription directed by the AP-1site or the RSV promoter and enhancer were relatively unaffected (FIG.18). These results strongly indicate that NF-AT_(c) contributessubstantially to IL-2 gene expression in T cells.

Dominant-negative NF-AT_(c) polypeptides or peptidomimetics thereof canbe used as pharmaceutical antagonists of NF-AT-mediated activation of Tcells. In one variation, such drugs can be used as commercial researchreagents for laboratory testing and analysis of T cell activation andthe like, among many other uses (e.g., immunosuppressant).

Example 15 Post-Translational Modification of NF-AT_(c)

Post-translational modification of NF-AT_(c) was investigated in cellstreated with agents that activate PKC or increase intracellular Ca++.Cells were transfected with NF-AT_(c) as described in FIG. 13 andstimulated as shown for 2 hrs plus or minus 100 ng/ml CsA. Whole celllysates were analyzed by western blotting as in FIG. 13. The bulk ofNF-AT_(c) in cells treated with ionomycin migrates faster than that innon-treated cells and this mobility shift is inhibited by CsA (FIG. 19,lanes 1, 3-4). This is consistent with a dephosphorylation event,possibly by direct action of calcineurin (Clipstone and Crabtree (1992)Nature 357: 695-697), however, any of a large number of processes couldproduce the observed mobility changes. There is evidence that NF-AT_(p)is a substrate for calcineurin, however, the mobility shifts produced byphosphatase treatment of NF-AT_(p) or NF-AT_(c) are far greater thanthose observed in FIG. 19. These observations indicate that NF-AT_(c) isnot a direct substrate of calcineurin. PMA treatment produces a slowermigrating NF-AT_(c) (FIG. 19, lane 2); therefore, PKC-activated pathwayslikely contribute to NF-AT activity by modification of NF-AT_(c) inaddition to activation of the nuclear component.

Equivalents

Although the present invention has been described in some detail by wayof illustration for purposes of clarity of understanding, it will beapparent that certain changes and modifications may be practiced withinthe scope of the claims.

1. A method for identifying an immunosuppressive agent, comprising: (i)providing a cell containing NF-ATc polypeptide which is encoded by anucleic acid that hybridizes under conditions of 5×SSc at 42° C. to SEQID NO:45 and has one or more of the following biological activities: (a)binds calcineurin; (b) undergoes nuclear localization upon T cellactivation; and (c) activates gene transcription upon T cell activation;(ii) contacting the cell of (i) with a compound that induces nucleartranslocation of the NF-ATc polypeptide; (iii) contacting the cellbefore, during or after step (ii), with a test agent; and (iv) assayingfor nuclear translocation of the NF-ATc polypeptide, wherein aninhibition of nuclear transport in the cell relative to a cell that wasnot contacted with the test agent indicates that the test agent is acandidate immunosuppressive agent.
 2. The method of claim 1, wherein theassaying for nuclear translocation comprises determining the nuclearpresence of NF-ATc polypeptide.
 3. The method of claim 1, wherein theassaying for nuclear translocation comprises determining the nuclearassociation between NF-ATc polypeptide and an NF-ATn polypeptide.
 4. Themethod of claim 1, wherein the assaying for nuclear translocationcomprises determining the binding of NF-AT polypeptide or anNF-ATc:NF-ATn polypeptide complex to an NF-AT DNA binding sequence. 5.The method of claim 4, comprising using a gel mobility shift assay todetermine the binding of the NF-AT polypeptide or an NF-ATc:NF-ATnpolypeptide complex to an NF-AT DNA binding sequence.
 6. The method ofclaim 1, further comprising determining the level of expression of atest nucleic acid linked to an NF-AT DNA binding sequence.
 7. The methodof claim 1, wherein the compound of step (ii) stimulates Ca++ release inthe cell.
 8. The method of claim 7, wherein the compound is ionomycin.9. The method of claim 1 wherein the cell further comprises an NF-ATnpolypeptide and wherein assaying for nuclear translocation includesdetermining the level of NF-AT complex comprising NF-ATc and NF-ATn,wherein the presence of a lower level of NF-AT complex relative to acell that has not been contacted with a test agent indicates that thetest agent is a candidate immunosuppressive agent.
 10. The method ofclaim 9 wherein assaying for nuclear translocation includes determiningthe level of NF-AT complex bound to an NF-AT binding sequence, whereinthe presence of a lower level of bound NF-AT complex relative to that ina cell that has not been contacted with the test agent indicates thatthe test agent is a candidate immunosuppressive agent.
 11. A method foridentifying an immunosuppressive agent, comprising (i) contacting apurified NF-ATc polypeptide or cell extract containing an NF-ATcpolypeptide with a purified NF-ATn polypeptide, or a cell extractcontaining an NF-ATn polypeptide and a test agent, under conditionswhich permit the formation of an NF-AT complex comprising NF-ATc andNF-ATn, wherein the NF-ATc polypeptide is encoded by by a nucleic acidthat hybridizes under conditions of 5×SSC at 42° C. to SEQ ID NO:45 andhas one or more of the following biological activities: (a) bindscalcineurin; (b) undergoes nuclear localization upon T cell activation;and (c) activates gene transcription upon T cell activation; and (ii)determining the level of NF-AT complex formed, wherein a lower level ofNF-AT complex relative to the level of NF-AT complex formation in theabsence of the test agent indicates that the test agent is a candidateimmunosuppressive agent.
 12. The method of claim 11, wherein the NF-ATcor NF-ATn polypeptide is immobilized.
 13. The method of claim 1 whereinthe cell further includes an NF-AT regulated enhancer region linked to atest nucleic acid; and assaying for nuclear translocation includesdetermining the level of expression of the test nucleic acid, wherein alower level of expression of the test nucleic acid relative to its levelof expression in a cell that was not contacted with the test agentindicates that the test agent is a candidate immunosuppressive agent.14. The method of claim 13, wherein the test gene encodes a proteinwhich is essential for cell proliferation or viability.
 15. The methodof claim 1 wherein the cell further includes an NF-AT regulated enhancerregion linked to a test nucleic acid; and assaying for nucleartranslocation includes determining the level of expression of the testnucleic acid, wherein a higher level of expression of the test generelative to its level of expression in a cell that was not contactedwith the test agent indicates that the test agent is a candidateimmunostimulatory agent.
 16. The method of claim 15, wherein the testnucleic acid encodes a protein which is essential for cell proliferationor induces cell death.
 17. A method for identifying an immune regulatingagent, comprising (i) contacting a cell or a cell extract containing anNF-ATc polypeptide with a test agent, wherein the NF-ATc polypeptide isencoded by a nucleic acid that hybridizes under conditions of 5×SSC at42° C. to SEQ ID NO:45 and has one or more of the following biologicalactivities: (a) binds calcineurin; (b) undergoes nuclear localizationupon T cell activation; and (c) activates gene transcription upon T cellactivation; and (ii) determining the level of phosphorylation of theNF-ATc polypeptide, wherein a difference in the level of phosphorylationrelative to that of a cell or cell extract that was not contacted withthe test agent indicates that the test agent is a candidate immuneregulating agent.
 18. The method of claim 17, further comprisingcontacting the cell with an agent which induces the nucleartranslocation of the NF-ATc polypeptide.
 19. A method of any one ofclaims 1, 9, 10, 13, 15, and 17, wherein the NF-ATc polypeptide isencoded by a heterologous nucleic acid in the cell.
 20. A method ofclaim 19, wherein the NF-ATc polypeptide or portion thereof comprises atleast 25 amino acids having an amino acid sequence which issubstantially identical to an amino acid sequence e set forth in SEQ IDNO:46.
 21. A method of claim 19, wherein the NF-ATc polypeptide orportion thereof is encoded by a nucleic acid which hybridizes to anucleic acid having the nucleotide sequence set forth in SEQ ID NO:45 orthe complement thereof.